Radical mechanisms of S-adenosylmethionine-dependent enzymes

RADICAL MECHANISMS OF S-ADENOSYLMETHIONINE-DEPENDENT ENZYMES BY PERRY A. FREY* AND SQUIRE J. BOOKER* *University of Wisconsin-Madison, Madison, Wisconsin 53705, and tDepartment of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802 I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1I. Lysine 2 , 3 - A m i n o m u t a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M o l e c u l a r P r o p e r t i e s a n d C o f a c t o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. T h e Ro le o f SAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Lysyl F r e e R a d i c a l s a n d t h e R o l e o f PLP . . . . . . . . . . . . . . . . . . . . . . . . . . . . I). T h e 5 ' - D e o x y a d e n o s y l R a d i c a l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. R e v e r s i b l e C l e a v a g e of SAM by [4Fe-4S] l+ . . . . . . . . . . . . . . . . . . . . . . . . . . III. Pyruvate F o n n a t e - L y a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M o l e c u l a r P r o p e r t i e s a n d R e a c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Py ru vate F o r m a t e - L y a s e A c t i v a t i n g E n z y m e , a n d t he R ol e of SAM . . . . . . . C. C h a r a c t e r i z a t i o n o f PFL-Activase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. A n a e r o b i c R i b o n u c l e o t i d e R e d u c t a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M o l e c u l a r P r o p e r t i e s a n d R e a c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. C h a r a c t e r i z a t i o n o f t h e Activase S u b u n i t a n d t h e Role of SAM . . . . . . . . . . V. Biotin S y n t h a s e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. B i o t i n S y n t h a s e R e a c t i o n a n d M o l e c u l a r P r o p e r t i e s . . . . . . . . . . . . . . . . . . B. C h a r a c t e r i z a t i o n of t h e I r o n - S u l f u r C l u s t e r s o f tim bioB P r o t e i n . . . . . . . . . C. N a t u r e o f t h e Sulfur D o n o r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. L i p o i c A c i d B i o s y n t h e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ret~wences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

! ,7 2 5 7 13 l6 19 IS) 21 24 26 26 30 32 32 36 37 38 12

I. I N T R O I ) U C T I O N

A family of novel S-adenosylmethione (SAM)-dependent enzymes has e m e r g e d in which SAM appears to serve as a source of 5'-deoxyadenosine-5'-yl, the 5'-deoxyadenosyl free radical. O n e m e m b e r of the family, lysine 2,3-aminomutase, makes use of the 5'-deoxyadenosyl radical to initiate the molecular r e a r r a n g e m e n t of a substrate, a n d the radical is subsequently r e g e n e r a t e d in the catalytic cycle, so that SAM functions in a catalytic role as a true coenzyme. The other family m e m b e r s use SAM as a substrate, a n d the 5'-deoxyadenosyl fi'ee radical appears to be an i n t e r m e d i a t e in an irreversible h y d r o g e n abstraction reaction. T h e latter enzymes include the pyruvate formate-lyase activating enzyme, the anaerobic ribonucleotide reductase from Escherichia coli, biotin synthase, a n d lipoic acid synthase.

AI)I~L",:(~5 1,% I'I~07EIN CttlfMI,W'RE lbl. 58

(;op~rigiat © 200i by A(admni( Ih('s~ All ligl3ts ot ]cpr,)du('ticm in ally torm testa ~('d I)0(15-327,3 q)l S~' ()l)

2

PERRYA.FREYANDSQUIREJ. BOOKER

The 5'-deoxyadenosyl radical is far from unique to SAM-dependent enzymes. It has long been regarded as the raison d'etre for the existence of adenosylcobalamin, the vitamin B12 coenzyme. Photolytic or thermal cleavage of adenosylcobalamin produces cob(II)alamin and the 5'deoxyadenosyl radical through homolytic scission of the Co-C bond. Moreover, spectral and kinetic evidence indicate that this same cleavage is brought about at the active sites of adenosylcobalamin-dependent enzymes, and the ensuing 5'-deoxyadenosyl radical is thought to initiate the free radical-based reaction mechanisms catalyzed by those enzymes. The apparently similar role of SAM in the reaction of lysine 2,3-aminomutase led to the designation of this coenzyme as "a poor man's adenosylcobalamin" (Frey, 1993; Frey et al., 1998), an appellation originally adopted by H. A. Barker. In this article we describe the family of SAM-dependent enzymes that make use of the 5'-deoxyadenosyl radical, with special reference to the mechanism by which SAM is cleaved reversibly at the active site. We also consider the functions of the 5'-deoxyadenosyl radical and the mechanisms of these diverse reactions.

II. LYSINE 2,3-AMINOMUTASE

A. Molecular Properties and Cofactors

This enzyme from Clostridium subteqminale SB4 was the first m e m b e r of the SAM family to be discovered. Barker and co-workers described it in 1970 as a hexameric enzyme (Mr 285,000; subunit Mr 47,000) that catalyzed the reversible transformation of L-lysine into L-~l-lysine according to Eq. ( 1 ) (Chirpich et al., 1970; Zappia and Barker, 1970). *

H

foo-

R" C" IOw~H NH3+

g,. "-

~"

~,C-C +H31~ COO-

R = +H~NCHzCHzCH2 (1)

The overall molecular weight and subunit composition were confirmed by light scattering and cross-linking experiments, respectively (Song and Frey, 1991). The interconversion of lysine into ~-lysine is analogous to adenosylcobalamin-dependent r e a r r a n g e m e n t reactions in which a hydrogen atom exchanges positions with a group b o n d e d to an adjacent carbon according to Eq. (2)(Frey, 1990).

S-ADENOSYLMETH1ONINE-DEPENDENT ENZYMES

i

I

__C~__C~__

~

I

__C~

'.{

[

C~__

I

i

J

I

X

H

H

X

X = CH2COOH, COSCoA, OH, NH2, etc. I

NH2

The reaction of lysine 2,3-aminomutase follows this pattern in every respect. The transferred hydrogen is not exchanged with solvent protons in the course of its migration; moreover, a similar amino group transfer is f o u n d in at least two adenosylcobalamin-dependent reactions. Until the discovery of lysine 2,3-aminomutase (LAM) by Barker and co-workers, all reactions of this type had been thought to require adenosylcobalamin, which itself had b e e n discovered by Barker and his associates (Barker et al., 1960). However, the coenzymes for lysine 2,3-aminomutase were f o u n d to be novel for this type of reaction and did not include adenosylcobalamin. The coenzymes included SAM, pyridoxal 5' -phosphate (PLP), and iron. The enzyme contains a b o u t one tightly b o u n d PLP per subunit (Song and Frey, 1991), and addition of PLP modestly stimulates activity. The enzyme preparation described by Barker and co-workers required addition of SAM to activate the enzyme. More recently, with purification u n d e r strictly anaerobic conditions inside an anaerobic chamber, samples of enzyme have been f o u n d to display significant activity without the addition of SAM, although activity was stimulated by SAM. The r e q u i r e m e n t for PLP a d d e d a n o t h e r e l e m e n t of novehy, in that PLP was known to facilitate carbanion formation in enzymatic reactions of amino acids, and this inevitably led to the incorporation of solvent hydrogen stably b o n d e d to carbon atoms of substrates and products. The absence of solvent-derived hydrogen in [3-1ysine was paradoxical for a P L P - d e p e n d e n t enzyme. Barker and co-workers originally r e p o r t e d the presence of iron in lysine 2,3-aminomutase (Chirpich et al., 1970); this was later found to be associated with a [4Fe-4S] cluster (Petrovich et al., 1991, 1992). Purification u n d e r strictly anaerobic conditions inside an anaerobic c h a m b e r dramatically increased the iron and sulfide content, as well as the enzymatic activity. The iron-sulfur cluster has been observed in [k)ur different forms, only lwo of which are relevant to catalysis (Petrovich et al., 1992; Lieder et al., 1998). Two of the iron-sulfur clusters are apparently artifacts of sensitivity to dioxygen in the a i r - when air is rigorously excluded during enzyme purification, these

4

PERRY A. FREYAND SQUIRE J. BOOKER

[0] [3Fe-4S]

[4Fe-4SI a+ -

g =2.015

g = 2.007

[H], AdoMet

\

-

[4Fe--4S] 2+ silent

"~

[4Fe--4s]l÷/AdoMet g = 1.96

RSH/Fe(II) FIG. 1. Four species ofiron-sulfllr cluster observed in LAM. In LAM purified inside an anaerobic chamber, the principal species of iron-sulfur center observed are shown as [4Fe-4S] 3+ a n d the EPR-silent [4Fe-4S] 2+. T h e more anaerobic the conditions of purification, the less 3+ form observed. Controlled oxidation of these species generates the [3Fe--4S]-cluster, which has b e e n characterized by EPR. This process is reversed by treatm e n t with a sulfhydryl reducing a g e n t a n d ferrous iron. Reduction of the 2+ species to [4Fe-4s] + requires b o t h a strong reducing agent, such as dithionite, a n d either SAM or SAH. T h e complex of LAM with [4Fe-4S] +, SAM, a n d substrate leads to the cleavage of SAM into m e t h i o n i n e a n d the 5'-deoxyadenosyl radical, which initiates the isomerization of lysine by abstracting a hydrogen atom from C3.

two forms are not observed in significant amounts. The interconversions of the iron-sulfur centers in their various observable states are summarized in Fig. 1. The enzyme purified u n d e r good anaerobic conditions contains a mixture of the forms designated as [4Fe-4S] 3+ and [4Fe-4S]2+. The f o r m e r is an inactive species that has not been fully characterized but that can be converted into the latter, an active form. These species were distinguished by electron paramagnetic reso n a n c e (EPR) spectroscopy, the inactive form [4Fe-4S] ~+ being characterized by its signal at g 2.007 and the active form [4Fe-4S] 2+ by its EPR silence. That these were four i r o n - f o u r sulfur clusters was confirmed by controlled oxidation to the three i r o n - f o u r sulfur species [3Fe-4S] +, which was characterized by EPR (g = 2.15) (Petrovich et al., 1992). The three i r o n - f o u r sulfur cluster can be transformed into the active [4Fe-4S] 2+ cluster by addition of ferrous sulfate and a reducing agent such as dihydrolipoate. The second active form of the iron-sulfur cluster results from the SAM-dependent reduction of the form [4Fe-4S] 2+ by dithionite. The reduction by dithionite does not proceed in the absence of SAM, although the analog S-adenosylhomocysteine (SAH) also potentiates this process (Lieder et al., 1998). The iron-sulfur cluster reduced u n d e r these conditions has been characterized as [4Fe-4S] 1+ (g = 1.96). The [4Fe-4S] 1+ cluster appears to be the form that potentiates the reaction of SAM as a coenzyme for lysine 2,3-aminomutase, and the mechanism of this process will be dealt with in a later section.

5kADENOSYLMETHIONINE-DEPEN DENT ENZYMES

B. The Role of S A M

In order to examine the hypothesis that the 5'-deoxyadenosyl moiety of SAM functions in the same capacity as in adenosylcobalamin-dependent reactions, the experimental tests documenting its role in mediating hydrogen transfer in Bl,2-rearrangements were applied to SAM in the reaction of lysine 2,3-aminomutase (Moss and Frey, 1987; Baraniak et al., 1989). LAM was activated with [5'-'~H]SAM and used to catalyze the transformation of lysine into its equilibrium mixture of lysine with [3-lysine, leading to the observation that both substrate and product were labeled with tritium [Eq. (3) ]. Lysine + [5'-3H]SAM

LAM > [~H]Lysine + [3-[:~H]Lysine + SAM (3)

It was found that all of the tritium in nonstereospecifically labeled [5' - 3H]SAM could be transferred to the substrate and product when the enzyme was present at less than 1:1 stoichiometry with the coenzyme, proving that both of the hydrogens on the 5'-methylene carbon of SAM participated in hydrogen transfer (Baraniak et al., 1989). This meant, as it did in the case of [5' - 3H]adenosylcobalamin in Bl2-dependent rearrangements, that the 5'-deoxyadenosyl moiety must have been cleaved from SAM at the active site, and the resulting nucleoside species must have mediated the hydrogen transfer process in the isomerization of lysine and [~-lysine. In contrast, the abstraction of deuterium from C3 of lysine was found to be stereospecific (R-), as was the incorporation of deuterium at C2 of ~-lysine (R-) (Aberhart et al., 1983). Transfer of tritium from [3-~H]lysine to SAM was also observed (Kilgore and Aberhart, 1991). Further e~fidence of the parallelism between SAM and adenosylcobalamin was provided by the demonstration that the conversion of lysine into ~-lysine by LAM proceeded with both intramolecular and intermolecular transfer of hydrogen. It was first shown by transformation of a mixture of lysine and lysine-3-d2 that hydrogen transfer in the rearrangement was intermoleculai; because the principal product was ~-lysine-dl when lysine was in large excess over the deuterated substrate (Aberhart et al., 1983). When the ratio of lysine to lysine-3-32 was varied over a substantial range, mass spectrometric analysis of the ~-lysine produced showed conclusively that [3-1ysine-d~2 was produced at low ratios of lysine with lysine-cb2 (Baraniak et al., 1989). Therefore, intramolecular transfer occurred in 11% of the enzymatic turnovers, the low percentage being attributable to statistical factors and to the primary kinetic isotope effects in the reaction of lysine(t,2. A plot of the fractional ~lysine-cb2/~-lysine-d 1 in the product against the

6

PERRYA. FREYAND SQUIREJ. BOOKER 0.3

0.2

~ 0.1

0.0

,

0

I

10 Lys-d2fl-~ys

,

I

20

FIG. 2. Intermolecular and intramolecular hydrogen transfer by LAM, The LAM reaction was conducted using mixtures of L-lysine and L-lysine-3-d2in various ratios as the substrates. The ~-lysine produced was purified and analyzed for the ratio of dideutero- to monodeutero-~3-1ysine. The ratios are plotted here and show that as the ratio of dideuterolysine to lysine in the substrate approaches zero, the ratio of dideutero-~-lysine to monodeutero-[]-lysine approaches 0.11. Thus, 11% of the deuterium transfers must be intramolecular.

ratio lysine/lysine-3-d2 in the substrate is shown in Fig. 2. T h e intersection o n the o r d i n a t e clearly shows that 11% o f the turnovers result in intramolecular transfer o f d e u t e r i u m . T h e simplest a n d m o s t obvious r a t i o n a l e o f the tritium a n d deut e r i u m t r a n s f e r e x p e r i m e n t s was t h a t t r a n s i e n t cleavage o f a 5'd e o x y a d e n o s y l m o i e t y f r o m SAM allowed it to m e d i a t e h y d r o g e n transfer, p r e s u m a b l y t h r o u g h t h e i n t e r m e d i a t e f o r m a t i o n o f 5'd e o x y a d e n o s i n e . T h e f o r m a t i o n o f 5 ' - d e o x y a d e n o s i n e in the catalytic cycle was verified (Moss a n d Frey, 1990). Because the 5 ' - d e o x y a d e n o s y l radical was the m o s t likely species to m e d i a t e h y d r o g e n transfer, it was p o s t u l a t e d t h a t SAM was s o m e h o w reversibly cleaved to this radical at the active site. T h e 5 ' - d e o x y a d e n o s y l radical c o u l d t h e n abstract the ~ H (3-pro-R) f r o m lysine b o u n d as a n a l d i m i n e to PLP to f o r m 5'd e o x y a d e n o s i n e a n d a lysyl radical as illustrated in Fig. 3. I s o m e r i z a t i o n

S-ADENOSYLMETHIONINE-DEPEN DENT ENZYMES

Ado-CH Met[ P=Lys-H~

.

.

.

.

I a°-e"3

. la oc., Meal

~PLP=-Lys •

-

] PLP=tB-Lys .

7

I ao-C.2 Me, I -

I pLp=13_Lys_Hc~

FIG. 3. Role of putative 5'-deoxyadenosyl radical in the reaction of LAM. The 5'deoxyadenosyl radical g e n e r a t e d in the active site initiates the isomerization of lysine by abstracting the C3(H) from the pro-R position of lysine, which is b o u n d to the enzyme as its external aldimine with PER Isomerization of the PLP=lysyl radical leads to thc PLP=[Mysyl radical, Abstraction of a hydrogen atom from the methyl group of 5'deoxyadenosine leads to the external aldimine of [Mysine with PLP and the regeneration of the 5'-deoxyadenosyl radical.

of the lysyl radical to the [Mysyl radical would follow, and abstraction of a hydrogen atom from the methyl group of 5"-deoxyadenosine would produce the [Mysylaldimine of PLP and regenerate the 5'-deoxyadenosyl radical. Two steps in this mechanism would be chemically novel: the cleavage of SAM to form the 5'-deoxyadenosyl radical and the rearrangement of the lysyl radical. However, the role of hydrogen abstractor for the 5'-deoxyadenosyl radical would follow the precedent of adenosylcobalamin. C. Lysyl Free Radicals and the Ro& of PLP

A reasonable mechanism for the rearrangement of the lysyl radical to the ~-lysyl radical in Fig. 3 is provided by the chemistry of the external aldimine with PLP. This mechanism is shown in Fig. 4, where the unpaired electron in the lysyl radical pairs with a re-electron of the aldimine linkage to form the azacyclopropylcarbinyl radical, a quasisymmetric species that may reopen in the forward direction to the ~lysyl radical or in reverse to the lysyl radical. The mechanism is analogous to the well-known cyclopropycarbinyl radical rearrangernent (Kochi et al., 1969), with the substitution of nitrogen for carbon in the key ring position. The carbocyclic version is so fast that it has been adapted as a radical clock reaction (Griller and Ingold, 1980). A chemical precedent for molecules incorporating nitrogen in the key ring position is provided by the observation of the interconversion of the parent radicals in Eq. (4) at low temperatures (Danen and West, 1974).

~N- C H 2

•

~-

~ N

~CH2

(,4)

A m o r e closely related chemical c o u n t e r p a r t is that in Fig. 5, where abstraction of the b r o m i n e atom from N-benzylidene-9-bro-

8

PERRY A. FREY AND SQUIRE J. BOOKER

Ado-{~H 2 H

Ado-(~H2

HhCOO ~-Lys

+H3N(CH2)3 /\

H H

N

HC

//

Lys

"-...___./

[3-Lysyl aldimine

/

HhCO0 -

I

\ Ado--CH 3

+H3N(CH2)3

//N

H HC

-

"%

Lysyl aldimine

Ado-CH 3

H

+H3N(CH2)3 ~""COO

Ado--CH 3 H

+H3N(CH2)3H ~

[

COO

-

+H3N(CH2)3 ~

.gH -~'~COO

.~cH

N

Lysyl radical

"CH3

[3-Lysyl radical

FIG. 4. Isomerization of lysyl radicals in the active site of LAM. Abstraction of the 3pro-R(H) from the external aldimine of lysine and PLP by the 5'-deoxyadenosyl radical at the active site of LAM produces the lysyl radical and 5'-deoxyadenosine. The isomerization of the lysylradical most simply proceeds by radical addition to the imine linkage to form the azacyclopropylcarbinyl radical, a quasisymmetric species with an aziridine ring that can open in either the forward or reverse directions. In the forward direction, the 13-1ysylradical is formed. This is the product-related radical, which abstracts a hydrogen atom from the methyl group of 5'-deoxyadenosine to regenerate the 5'deoxyadenosyl radical while forming the aldimine of [Mysine with PLE Release of ~-lysine and binding of lysine in several steps recharges the enzyme for another isomerization cycle.

m o m e t h y l - D L - a l a n i n e ethyl e s t e r g e n e r a t e s a n a n a l o g o f the lysyl radical in Fig. 4. T h e m a i n p r o d u c t is t h e r e a r r a n g e d species, N-benzylidene-2-methyl-13-alanine ethyl ester, a r i s i n g f r o m q u e n c h i n g o f t h e r e a r r a n g e d radical, a n a n a l o g o f t h e ~-lysyl r a d i c a l in Fig. 4 ( H a n a n d Frey, 1990). T h i s m e c h a n i s m f o r t h e e n z y m a t i c r e a r r a n g e m e n t has b e e n established by d i r e c t o b s e r v a t i o n o f the ~-lysyl r a d i c a l a n d closely r e l a t e d a n a l o g s o f the lysyl radical in Fig. 4. O n m i x i n g lysine 2 , 3 - a m i n o m u tase with lysine a n d SAM a n d f r e e z i n g in the steady state with liquid N2, a s t r o n g signal at g = 2.0 a p p e a r e d in the E P R s p e c t r u m at 77°K

,%ADENOSYLMETHIONINE-DEPENDENT

BrA
ENZYMES

"-.
t}

~.

COOEt

(--.) It

It

y - Bu3SnH

-•COOEt 7%

~"~COOEt

93%

FIG. 5. A chemical model for the 2,3-aminomutation catalyzed by LAM. Treatment of the benzaldimine of 2-methyl-3-bromoalanine ethyl ester with the tributyltin radical, generated by reaction of tributyltin hydride with azo-bis-2-cyanopropionitrile, leads to abstraction of the bromine atom from C3. The resulting C3 radical is an analog of the lysyl radical in Fig. 4. Radical rearrangement and quenching leads to the isomerized product benzaldimine of 2-methylalanine ethyl ester.

(Ballinger et al., 1992a, 1992b). T h e signal was m a x i m a l in intensity at the initial steady state. W h e n the r e a c t i o n m i x t u r e was f r o z e n in liquid N2 at various times over the c o u r s e o f the r e a c t i o n , the intensity o f this signal gradually d e c r e a s e d to a lower e q u i l i b r i u m level at the same rate as the a p p r o a c h to c h e m i c a l e q u i l i b r i u m for free lysine a n d ~lysine. Moreover, the intensity o f the signal at the steady state was prop o r t i o n a l to the e n z y m e c o n c e n t r a t i o n . All o f these observations s u p p o r t e d the a s s i g n m e n t o f the EPR signal to an o r g a n i c free radical i n t e r m e d i a t e . T h e use o f [2-2H]lysine as the substrate dramatically n a r r o w e d the signal, a n d the use o f [2-13C]lysine dramatically b r o a d e n e d it, as shown in Fig. 6. Because the n u c l e a r h y p e r f i n e spin coupling c o n s t a n t f o r d e u t e r i u m with an u n p a i r e d e l e c t r o n in a ~-radical orbital is m u c h smaller t h a n that for h y d r o g e n , a n d the l~C-nuclear h y p e r f i n e spin c o u p l i n g c o n s t a n t is very large, the effects o f 2-2H a n d

10

PERRYA. FREYAND SQUIRE J. BOOKER

I

,

I

8150

•

,

.

,

!

3200

,

.

i

I

3250

,

.

,

I

•

I

3300

Ho (Gauss) FIG. 6. EPR spectra of the ~-lysylradical at the active site of LAM at 77°K. Spectrum a is that of LAM (30 to 35 ~tM) mixed with lysine and frozen at 77°IL Spectrum b is that obtained with the substitution of 1ysine-3,4,5,6-d8 for lysine. Spectrum c was obtained with the substitution of lysine-2-d] for lysine. Spectrum d was obtained with the substitution of [2-13C]lysine for lysine. Spectra were adapted from Ballinger et al. (1992b with permission of the American Chemical Society.

2-13C s u b s t i t u t i o n d e f i n i t i v e l y e s t a b l i s h e d t h e s t r u c t u r e as t h a t o f t h e IBlysyl r a d i c a l in Fig. 4. R a p i d m i x - f r e e z e q u e n c h E P R e x p e r i m e n t s p r o v e d t h e lysyl r a d i c a l to be kinetically competent as a n i n t e r m e d i a t e . T h e s i m p l e s t a p p r o a c h to a d d r e s s i n g t h e q u e s t i o n o f k i n e t i c c o m p e t e n c e w o u l d b e

,S-ADENOSYLMETHION1NE-DEPENDENT

ENZYMES

1l

to mix LAM with SAM and lysine in a rapid mixing device, q u e n c h with liquid isopentane at various times on the scale of milliseconds, and measure the signal intensity in the q u e n c h e d samples as a flmction of time to obtain the rate constant for appearance of the signal. This proved to be impractical because the activation by SAM took place on the time scale of seconds, not on the enzymatic turnover scale of milliseconds. Therefore, a n o t h e r approach to the question of kinetic c o m p e t e n c e had to be devised. The signal-broadening effect of 13C was adapted for this purpose. In initial experiments, LAM was mixed with SAM and a modest concentration of [2l:~C]lysine in a mixing device (Update Instruments) that allowed it m be held for 5 sec, allowing complete activation by SAM and signal development to reach its m a x i m u m , and then mixed a second time with a much larger concentration of unlabeled lysine. The reaction was freeze q u e n c h e d at various times on the millisecond time scale after the second mixing. The q u e n c h e d samples were analyzed bv EPR, and the signal u n d e r w e n t narrowing to that of the unlabeled lysyl radical on the millisecond time scale, consistent with the turnover n u m b e r of LAM (Ballinger, 1993). To d e t e r m i n e the turnove rate constant for the lysyl radical, this approach was adapted to the use of [2-2H]lysine for the initial mixing to develop the narrowed signal in Fig. 6, followed by a secondary mixing with an excess of unlabeled lysine to b r o a d e n the signal before q u e n c h i n g on the millisecond time scale (Chang et al., 1996). A plot of the fraction of unlabeled lysyl radical against time was fitted to a first-order rate equation, and the rate constant was 24 s-1 at 21°C, the same as the turnover n u m b e r kca t for LAM at this temperature. Therefore, the lysyl radical is kinetically c o m p e t e n t as an intermediate. Reed's method for enhancing the resolution of EPR spectra was adapted to a detailed analysis of the EPR signal for the ~-lysyl radical (Ballinger and Reed, 1995). The intensity of the ~-lysyl radical signal in the steady state was high enough that very little noise appeared in the spectra when LAM was used at concentrations of 30 to 50 btM. This allowed resolution enhancement of the spectral envelope to permit unmasking the primary underlying splittings. Computer simulations of the resolution enhanced spectra allowed the splitting constants to be estimated, both for hydrogen in the unlabeled lysyl radical and for the species with deuterium or 15N-labeling at C2 and C3-C6. From these splitting constants, and comparable literature data on model free radicals, the dihedral angles relating the C3-proton and C3-nitrogen to the radical orbital could be set at 70 ° and 10 °, respectively. These values fixed the conformation of the radical as N3-pyridoxylidene-~-lysine-2-yl shown below.

PERRYA. FREYANDSQUIREJ. BOOKER

12

H

+H3N(CH2)3~ " i ~ . . . . . ~ . H

2

~

14, ~

- OOC ~OH

~N"

~H 3

N~-5'-Phosphopyridoxylidene-13-1ysine-2-yl Although the EPR results placed the unpaired electron on the ~-lysyl carbon and nitrogen skeleton in the conformation shown above, they did not implicate PLP in the structure. This was done by electron spin echo envelope modulation (ESEEM) spectroscopy. [4'-2H]PLP was used in place of PLP with LAM and lysine to generate the free radical, and a freeze q u e n c h e d sample revealed a prominent doublet in the ESEEM spectrum centered at the Larmor frequency for deuterium. No such signal appeared in a matched sample prepared with PLP (Ballingeret al., 1995). This observation assured that the 4'-2H of PLP resided less than 6 ]k from the unpaired electron. From the physical characteristics of the doublet signal, the distance between the unpaired electron and the deuterium in [4'-2H]PLP was calculated as 3.4 A. This distance placed PLP within the required range for the external aldimine shown above, thus implicating PLP in the structure. The identification of the ~-lysyl radical strongly supported the free radical mechanism in Figs. 3 and 4. However, three other radicals participate in the mechanism in addition to the [3-1ysylradical: the lysyl and azacyclopropylcarbinyl radicals and the 5'-deoxyadenosyl radical. In the steady state and throughout the course of the reaction, only the I]-lysyl radical is observable by EPR spectroscopy with lysine as the substrate and SAM as the coenzyme. This fact does not exclude other radicals as participants in the mechanism because the [3-1ysylradical is the most stable radical among those in Fig. 4. The lysyl and 5'-deoxyadenosyl radicals lack the resonance delocalization of the [3-1ysyl radical, and the azacyclopropylcarbinyl radical incorporates the strained aziridine ring that would make it a high-energy species. These other radicals could be present in unobservable concentrations at the steady state. In order to observe them by EPR spectroscopy, stabilizing structural features that would increase their concentrations to or above that of the []-lysyl radical are required. Such stabilization for the lysyl radical is provided by

S-ADENOSYLMETH1ONINE-DEPENDENT ENZXJ%IES

1~{

the n o n b o n d i n g electrons on sulfur in 4-thia+-lysine, which reacts as a substrate for LAM according to Eq. (5) (Wu et al., 1995). J

NH3 +

[ +H3N ~ S H

NH3 + +

NIt++ +

(5~ tI~'-CO

O-

O

The 4-thialysyl radical is the dominant free radical at the active site of LAM in the steady state of the reaction of 4-thia-L-lysine. The EPR spectra of this radical, unlabeled and labeled with deuterium or carbon-13 at C3, are shown in Fig. 7. These spectra characterized the paramagnetic species as the 4-thia analog of the substrate-related lysyl radical in Fig. 4. The identification of the ~-lysyl radical as an intermediate of the reaction of lysine and of the 4-thia analog of the lysyl radical as an intermediate in the reaction of 4-thialysine strongly support the radical rearrangement mechanism in Fig. 4. PLP is not a new coenzyme; it is well known to facilitate carbanion chemistry of amino acids. However, its role in reaction of LAM is novel. While the formation of the external aldimine with lysine is conventional PLP chemistry, facilitation of radical isomerization by PLP is novel. PLP is likely to play a similar role in the adenosylcobalamin-dependent aminomutase reactions described by Stadtman (1973).

D. The 5"-Deoxyadenosyl Radical Lysyl free radical formation, as illustrated in Figs. 3 and 4, is initiated by the 5'-deoxyadenosyl radical, which arises from a reversible cleavage of SAM. This same radical has long been inferred to be a hydrogen abstracting intermediate in adenosylcobalamin-dependent reactions (Frey, 1990). However, it has never been observed spectroscopically, either in the reaction of LAM or in any Blz-dependent reaction. The absence of its signal from the EPR spectra of reaction mixtures in which it is thought to participate may be attributed to its high energy and fleeting existence. Thus, it is generally regarded as so reactive that it never exists at observable concentrations. In a sense, this property forms the foundation for its utility as a radical initiator that removes hydrogen atoms from unreactive carbon atoms of substrates. However, recognition of this essential property does not diminish the need for physical evidence of its actual existence and participation.

14

PERRYA. FREYANDSQUIREJ. BOOKER

ZR2

I

,

I

!

I

I

•

|

|

I

I

:3200

•

I

I

I

I

J

I

I

33oo

~SUSS FIG. 7. EPR spectra of the 4-thialysyl radical at the active site of LAM. The top spectrum is that of LAM mixed with 4-thia-L-lysine and frozen at 77°K. The middle spectrum was obtained with the substitution of 4-thialysine-3-d~2 for 4-thialysine. The lower spectrum was obtained with the substitution of 4-thia[3J3Cllysine for lysine. The spectra are taken from Wu et al. (1995) and reproduced with permission of the American Chemical Society.

A t t h e p r e s e n t t i m e , t h e s p e c t r o s c o p i c o b s e r v a t i o n o f a 5'deoxyadenosyl radical requires that some chemically stabilizing funct i o n a l g r o u p b e i n c o r p o r a t e d i n t o a d e r i v a t i v e t h a t w o u l d i n c r e a s e its s t a b i l i t y e n o u g h to a l l o w it t o b e o b s e r v e d . T h e d e r i v a t i v e m u s t b e a c c e p t e d a t t h e a c t i v e site o f a n e n z y m e ; t h a t is, it m u s t n o t i n c r e a s e t h e

S-ADENOSYLMETHIONINE-DEPENDENT

ENZYMES

l ~)

steric requirements of the coenzyme or introduce electrostatic charges that might be repelled by the active site. At the same time, the very stability of the derived radical analog would diminish its effectiveness as an initiator of radical-based mechanisms. Such an analog of SAM is 3',4'anhydroadenosyl-L-methionine (anSAM) shown below (Magnusson et al., 1999). NH2

S +-

CH

-OOC~/~ NH3 +

OH

3',4"-Anhydroadenosylcobalamin (anSAM)

Homolytic scission of the S-anhydroadenosyl b o n d would lead to the 5'-deoxy-3',4'-anhydroadenosyl radical, an allylic analog of the 5'deoxyadenosyl radical. The allylic analog should be stable enough to be observed by EPR spectroscopy. anSAM activates LAM to about 0.5% of the activity elicited by SAM. Moreover, in the steady state of reactions of LAM with anSAM and lysine the only free radical that is detected by EPR is that of the 5'deoxy-3',4'-anhydroadenosyl radical. The EPR signal of this radical is shown in Fig. 8, together with the signals resulting from substitution of deuterium on C5' and of lsC for all of the ribosyl carbons. These signals characterize the species as the 5'-deoxy-3',4'-anhydroadenosyl radical. Kinetic studies have shown that this radical is formed at a faster rate than the overall isomerization reaction activated by anSAM (unpublished), showing that it is kinetically competent as an intermediate. EPR spectroscopic experiments verify that it is labeled with deuterium when it activates LAM in the isomerization of [3-2H2]lysine, proving that it mediates hydrogen transfer. The only species of the 5'-deoxyadenosyl radical to be observed to date is the allylic 3',4'-anhydro derivative. It functions as a true coenzyme in the reaction of LAM in the same way as the putative 5'deoxyadenosyl radical. The fact that anSAM is less effective in activating LAM than SAM does not diminish the case for the 5'-deoxyadenosyl radical, because the allylic analog must be expected to be less reactive. All things considered, the observation of the 5'-deoxy-3',4'-anhydroadenosyl radical strongly supports the case for the participation of the 5'-deoxyadenosyl radical in the SAM activation of LAM.

16

PERRYA. FREYAND SQUIRE J. BOOKER

A

B

C

D

I

I

I

I

I

I

300O

3100

3200

33OO

340O

3500

Gauss

FIG. 8. EPR spectra of the 5'-deoxy-3',4'-anhydroadenosyl radical at the active site of LAM. The spectrum in (A) was obtained with LAM, L-lysine, and anSAM and is that of the 5'-deoxy-3",4'-anhydroadenosylradical in the steady state of the reaction after freezing at 77°IL The spectra of (B) and (C) were obtained by substituting L-lysine-3,4,5,6-d8 for lysine. The radical was labeled with deuterium by the C3(D) of the substrate. The spectrum in (D) was obtained after substituting [l',2',3',4',5'-13C5]anSAMfor SAM. The spectra are reproduced from Magnusson et al. (1999) and used with permission from the American Chemical Society.

E. Revev'sible Cleavage of S A M by [4Fe-4S] 1+ T h e m o s t obvious m e a n s by which SAM c o u l d be cleaved reversibly to the 5 ' - d e o x y a d e n o s y l radical at an enzymatic site w o u l d be by reversible e l e c t r o n transfer to the c o e n z y m e . H o m o l y t i c cleavage to the nucleoside radical a n d a methionine-S-yl radical w o u l d r e q u i r e i n p u t o f the b o n d dissociation e n e r g y o f m o r e t h a n 60 kcal mo1-1. A m e c h a n i s m for the transfer o f an e l e c t r o n to SAM entails sufficient m e c h a n i s t i c p r o b lems, b u t w o u l d avoid the f o r m a t i o n o f two h i g h - e n e r g y radicals. In LAM, an obvious s o u r c e o f an e l e c t r o n for this p u r p o s e is the iron-sul-

,~ADENOSYLMETHIONINE-DEPENDENT ENZYMES

17

fur cluster in its most highly reduced form, [4Fe-4S] I+/SAM shown in Fig. 2. This complex can be formed by reduction of the EPR-silent [4Fe-4S] 2+ with dithionite, but only in the presence of SAM (Lieder et al., 1998). This reduction is an absolutely essential step in the activation of LAM. Once the reduction to the complex LAM/[4Fe-4S]I+/SAM is complete, the enzyme is fully active and dithionite is no longer required for activity. The SAM-analog SAH also potentiates this reduction, although SAH does not serve as a coenzyme for I2kM in the catalytic reaction. In the absence of SAM or SAH, dithionite does not reduce [4Fe-4S] 2+ at the active site. Clearly, binding interactions between SAM (or SAH) and LAM modulate the reduction potential of [4Fe-4S] 2+ to a less negative value and allow the enzyme to be activated. An electron transfer within the reduced complex could provide for the reversible formation of methionine and the 5'-deoxyadenosyl radical according to Eq.(6). If this were to take place in a protected site, where the radical could be sequestered from adventitious oxidizing or reducing agents and directed to subsequent reaction exclusively with lysine or ]]-lysine, the highly reactive hydrogen-abstracting 5'deoxyadenosyl radical could fhnction efficiently.

(~) HO

OH

HO

OH

The essential role of the iron-sulfur center [4Fe-4S] ~+ is to transfer an electron to SAM, but the mechanism of this process has not been known. Four mechanisms that have been suggested in c o n n e c t i o n with studies of LAM, pyruvate formate lyase, and the anaerobic ribonucleotide reductase of E. coli are illustrated in Fig. 9. In mechanism a, the iron-sulfur center simply transfers an electron to SAM, and the resulting radical undergoes a fragmentation to m e t h i o n i n e and the 5"-deoxyadenosyl radical. This mechanism would require the iron-sulfur center to display an exceedingly negative reduction potential, owing to the difficulty of transferring an electron to the sulfur of SAM, This sulfur has an inert gas electronic configuration, so that an additional electron would have to be inserted into a higher q u a n t u m level. In any case, it is not clear how an enzyme could control the f r a g m e n t a t i o n of a SAM radical and prevent the formation of the methyl or methionyl radical. Mechanism b overcomes the prob-

18

PERRYA. FREYAND SQUIREJ. BOOKER

.S--Cys S_Fe2+ S / Sj

Ado I CH2 "S+-CH3 Met /

Cys Ado, ,,S CH 2 S--Fe 2+ 'S+- CH3 S / S} Met/.

.S-.Cys r S~Fe3+ S S]

~

Cys Ado .S*'-'-CH2 S --Fe 2+ S --CH s S/SI Met /

Ado S~Fe 2÷ S S~

I CH2 ~S+--CH3 Met/

Ado f CH2 "S'---CH3 Met /

.S--Cys S~Fe2. S S]

Cys Ado S • CH2 S --Fe 3+ S -- CH 3 S / S[ Met /

..Ado -

CH2 /4+ S--Fe S/SI

H3Cx +/CH2-Ado ..-S

•

S--CH 3 Met/

H31C S/JS

Ado .~H2 /S--CH 3 Met

tAdo CH2

S~Fe 3+ S SI

/S--CH 3 Met

• CH2-Ado

Met

FIG. 9. Four mechanisms for the reversible cleavage of SAM by [4Fe--4S] 1+. Mechanisms a to d have been postulated for the cleavage of SAM by reaction with the [4Fe-4S]-cluster in LAM or in other enzymes in the family of enzymes that use SAM as a radical initiator. Long-range or second-sphere electron transfer from the iron-sulfur center in mechanism a to SAM produces a SAM radical. Fragmentation of the SAM radical leads to the 5'-deoxyadenosyl radical. In mechanism b, SAM acts as an alkylating agent that adenosylates a sulfur in the iron-sulfur center. Fragmentation of the adenosylated iron-sulfur center leads to the 5'-deoxyadenosyl radical. In mechanism c, SAM alkylates an iron in the iron-sulfur center, and fragmentation of the Fe-C5' bond generates the 5'-deoxyadenosyl radical. According to mechanism d, a coordination position of an iron in the iron-sulfur center is available for interaction with the nonbonding electron pair on the sulfur in SAM. Concerted, inner-sphere electron transfer and cleavage of the S-C5' bond leads to the 5'-deoxyadenosyl radical. Available evidence favors mechanism d, in which SAM is required to reduce the iron-sulfur center to [4Fe-4S] + (Lieder et al., 1998), and the presence ofa substrate is required for the concerted electron transfer and cleavage of the S-C5' bond. Cleavage of this bond is associated with direct ligation of the sulfur of methionine with iron (Cosper et al., 2000).

l e m o f c o n t r o l l i n g f r a g m e n t a t i o n b y p o s t u l a t i n g t h a t SAM, a n a l k y l a t ing agent, adenosylates a sulfur in the iron-sulfur center (Frey and R e e d , 1 9 9 3 ) . F r a g m e n t a t i o n w o u l d l e a d to t h e 5 ' - d e o x y a d e n o s y l r a d i cal a n d a o n e - e l e c t r o n o x i d i z e d c l u s t e r . E x t e n s i v e e x p e r i m e n t s i n s e a r c h o f e v i d e n c e s u p p o r t i n g this m e c h a n i s m f a i l e d to p r o v i d e a n y i n d i c a t i o n t h a t it c o u l d o p e r a t e .

S-ADENOSYLMETHIONINE-DEPENDENTENZYMES

19

Mechanisms c and d differ in that both require a direct coordination of SAM with an iron associated with the iron-sulfur centet: These interactions could take place either through a vacant ligand site in the center or by ligand exchange with an amino acid side chain, which may serve as a ligand in the absence of SAM. In mechanism c, the 5'-deoxyadenosyl moiety becomes ligated to iron transiently and, through homolytic scission of the Fe-C bond, enters into equilibrium with Fe 3+ and the 5"-deoxyadenosyl radical. In mechanism d, the sulfur of SAM is placed in a position allowing its nonbonding electron pair to coordinate weakly with iron. This contact is postulated to be brought about through binding interactions between LAM and SAM. In this mechanism, enzyme-SAM binding promotes an inner sphere electron transfer from the iron-sulfur center in concert with the cleavage of SAM into methionine and the 5'-deoxyadenosyl radical. Cleavage is coupled with an increase in the strength of the bonding between methionine with Fe ~+. The enzyme-SAM binding interactions control the regiospecificity of the cleavage of SAM, so that the 5'deoxyadenosyl radical is the only high-energy species generated. X-ray absorption spectroscopic experiments using Seadenosyl-selenoL-methionine (SeSAM), which activates tAM, supports mechanism d by showing a direct coordination between selenium and iron in complexes that include the 4,5-dehydrolysyl radical (Cosper et al., 2000). The Se-Fe coordination was also observed in a complex that included selenomethionine, 5'-deoxyadenosine, and LAM with the iron-sulfur center in the form of [4Fe-4S] 2+. The latter complex is regarded as structurally analogous to substrate-based radical complexes in the mechanism. The Se-Fe ligations in these complexes strongly support mechanism d in Fig. 9.

III. PYRUVATE FORMATE-LYASE

A. Molecular Properties and Reaction

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of pyruvate and coenzyme A (CoA) into acetyl-CoA and formate. Tile enzyme displays turnover numbers of 770 and 260 s-1 (30°C, p H 8.1) in the forward and reverse directions, and an equilibrium constant (K~@ of 750 for the overall reaction under the same conditions (Kessler and Knappe, 1996; Knappe et al., 1974, 1993). PFL is the anaerobic counterpart of the pyruvate dehydrogenase complex, and as such, it is one of three enzymes that are considered to be the foundation for the anaerobic life of E. coli and other organisms (Scheme l). Unlike the pyruvate

20

PERRYA. FREYANDSQUIREJ. BOOKER

pyruvate + CoA

~

a c e t y l - C o A + Pi

:~

acetyl-phosphate

+ ADP

~

1 2 3

-

acetyl-CoA + formate

-

acetyl-phosphate

~

acetate + ATP

+ CoA

SCHEME ]. The synthesis of ATP from pyruvate in anaerobically growing Escherichia coli. Reaction 1 is catalyzed by pyruvate formate-lyase. Reaction 2 is catalyzed by phosphotransacetylase. Reaction 3 is catalyzed by acetate kinase.

dehydrogenase complex, in which pyruvate is oxidatively decarboxylated with coupled reduction of NAD to NADH, the reaction catalyzed by PFL does not involve redox chemistry (Kessler and Knappe, 1996). Pyruvate formate-lyase is a homodimeric protein of Mr = 170,000, each polypeptide containing 759 amino acids (Conradt et al., 1984; R6del et al., 1988). It contains no metals or organic cofactors, and is synthesized in an inactive form (Ei). Conversion of the inactive form to the active form (Ea) is catalyzed by PFL-activase, and is dependent on SAM, pyruvate, and an electron source (Knappe et al., 1969). In vivo, the electron is supplied by NADPH via the flavodoxin/flavodoxin reductase reducing system. Other artificial one-electron donors are also effective, and 5-deazaflavin plus light, or dithionite are routinely employed (Conradt et al., 1984). Activation takes place only in the presence of pyruvate or its oxamate analog, which regulates the reaction allosterically (Knappe et al., 1974). A thorough kinetic investigation of the PFL reaction indicated that it follows a ping-pong mechanism with an acetyl-enzyme intermediate. Consistent with this mechanism, the enzyme catalyzes an exchange between formate and the carboxyl group of pyruvate (Knappe et al., 1974). Similarities with the (]3-keto)thiolase reaction of the fatty acid degradation cycle led to the suggestion that PFL might proceed by a similar mechanism (Knappe et al., 1974). This would involve attack of an enzyme amino acid side chain onto C2 of pyruvate, with concomitant displacement of formate. The resulting enzyme-bound acetyl group would be subsequently transferred to CoA in a second nucleophilic displacement. The authors duly noted the lack of precedent in the organic chemistry literature for cleaving the C2-C1 bond of pyruvate in this fashion, as well as the difficulty associated with rendering formate nucleophilic in the reverse reaction (Knappe et al., 1974). Indeed, the cleavage is now attributed to one-electron chemistry rather than two-electron chemistry. The

5-ADENOSYLMETHIONINE-DEPENDENT ENZYMES

2]

mechanism of PFL action will not be addressed in this review. Several excellent reviews have b e e n written on the subject (Knappe et al., 1993; Stubbe and van der Donk, 1998), including Knappe and Wagner's chapter in this volume. New insight has e m e r g e d from the three-dimensional structure of the enzyme (Becket et al., 1999), as well as recent biochemical studies with the mechanism-based inhibitor methacrylic acid (Plaga et al., 2000). B. Pyruvate Formate-Lyase Activating Enz~,me, and the Role of SAM A major achievement in the understanding of the mechanism of PFL was the elucidation of the nature of the conversion of E1 to E~,. Results of studies with radiolabeled forms of SAM indicated that it functioned at a chemical level rather than as an allosteric effector. In the presence of pyruvate, reduced flavodoxin, PFL-Ei, and partially purified PFL-activase, SAM was converted into methionine, adenine, and 5-deoxyribose concomitant with generation of PFL-Ea (Knappe and Schmitt, 1976). Subsequent controls indicated that adenine and 5-deoxyribose were the result of contaminating nucleosidase activity, and that methionine and 5'-deoxyadenosine were the true products of the reaction (Knappe and Schmitt, 1976; Knappe et al., 1984). The failure of any portion of SAM to co-migrate with Ea on activation and gel filtration indicated that activation was not due to modification of PFL by the molecule (Knappe and Schmitt, 1976). The stoichiometry of the activation reaction with respect to SAM (in which a hydride equivalent is transferred to its 5'-deoxyadenosyl moiety), its d e p e n d e n c e on reduced flavodoxin, and the enhanced sensiti> ity of E~ to oxygen, were suggestive of a redox process involving oxidation of PFL-Ei. Subsequent EPR investigations of PFL u n d e r strict anaerobic conditions revealed that activation of the enzyme coincided with formation of a paramagnetic species on the protein (Knappe et al., 1984). The EPR spectrum was centered at g= 2, and had a doublet splitting of 1.5 mT. The spectrum was abolished when the activated enzyme was exposed to oxygen, or hypophosphite, a mechanism-based inhibitor of the PFL reaction, In addition, hypophosphite covalently modified PFL-E~, but did not modifT PFL-Ei, or PFL-Ea that was previously inactivated with oxygen (Knappe et al., 1984). In the presence of active acetyl-PFL, quenching of the radical by hypophosphite at 0°C resulted in a new transient radical species (file = 20 rain at 0°C). Its spectral properties were consistent with a l-hydroxy-l-phosphow1 ethyl radical, thereby implicating the importance of the radical in catalysis (Unkig et al., 1989).

22

PERRYA. FREYAND SQUIRE J. BOOKER

O Cys41s

II

•

I

I

S--P--C--CH 3 O_ OH

1-hydroxy-l-phosphoryl ethyl radical adduct Subsequent labeling studies indicated that the radical was carbon centered, and that the hyperfine coupling was due to an exchangeable proton (Unkig et aL, 1989). Two additional experiments helped to elucidate the identity of the radical. The destruction of the radical by oxygen, which is accompanied by loss of enzyme activity, resulted in cleavage of the polypeptide chain to produce fragments of 82 and 3 kDa. The N terminus of the 3-kDa fragment was resistant to sequencing by Edman degradation, but analysis by mass spectrometry subsequent to trypsin digestion showed that the cleavage took place at glycine 734, which had been converted into an oxalyl group (Wagner et al., 1992). This corroborated labeling studies with auxotrophic strains of E. coli, in which administration of [2-13C] glycine and [1-1~C]glycine led to broadening of the radical signal when the spectra were acquired in D20. Simulation of various isotopically substituted forms of PFL showed approximately 55% of the spin density to be localized on C2 of glycine 734, with the remainder delocalized over the flanking carboxamide groups (Wagner et al., 1992; Knappe and Volker Wagner, 1995). The role of SAM in PFL activation was established from two different labeling studies that were analogous to the tritium transfer experiments carried out on lysine 2,3-aminomutase (Moss and Frey, 1987). In a study from the Kozarich laboratory, PFL was expressed in the presence of [22H]glycine stereospecifically labeled in either the pro-R or proS position, and then used to investigate the kinetics of the activation reaction. A "nominal" primary kinetic isotope effect was observed on the activation reaction only when deuterium was placed in the proS position (Wong and Kozarich, 1994). The Knappe laboratory using NMR spectroscopy and mass spectrometry (Frey et aL, 1994) demonstrated the transfer of deuterium from [2-2H]glycine to 5'-deoxyadenosine. In addition, peptides that were homologous to the Gly-734 sequence of PFL were found to act as competitive inhibitors of PFL activation, and stimulators of SAM cleavage. The peptide R-V-S-G734-Y-A-V, which encompassed amino acids 731-737 of PFL, supported cleavage of SAM to methionine and 5'-deoxyadenosine with Km and Vmaxvalues of 0.22 mM and 11 nmol rain -1 m g -1. Moreover, a similar peptide in which G734 was replaced with D-alanine displayed kinetic constants of 0.05 mM and 12 nmol min -1 m g -1. In contrast, when D-alanine was replaced

23

&ADENOSYLMETHIONINE-DEPENDENT ENZYMES

eH3CN + ~ S-

~

-O ~\

Methionine

_Ade

Ade PFL-activase HO

I OH

COO

O

+ "@H"

Q(o Ade

O SCHEME2, Trapping of a 5'-deoxyadenosyl radical by a dehydroalanyl-containing peptide. An octapeptide (Succ-Arg-Val-Pro-kAla-Tyr-Ala-Val-Arg-NH2)in which Gly-734 was altered to dehydroalanine (kAla), was used as a substrate tor PFL-activase. Incubation of the octapeptide with PFL-activase under turnover conditions resulted in adenosylation of the dehydroalanyl residue. The postulated mechanism inw)lves addition of a generated 5'-deoxyadenosyl radical to the C=C bond of the AAla, followed by quenching of the resulting backbone-centered radical.

with t.-alanine in the same peptide, it acted n e i t h e r as a stimulator o f SAM cleavage, n o r as an i n h i b i t o r o f the reaction. This is consistent with abstraction o f the pro-S h y d r o g e n o f PFL by a putative 5'd e o x y a d e n o s y l radical (Frey et al., 1994). Evidence in s u p p o r t o f an i n t e r m e d i a t e 5'-deoxyadenosyl radical was o b t a i n e d f r o m studies in which the activation r e a c t i o n was p e r f o r m e d with an o c t a p e p t i d e c o n t a i n i n g a d e h y d r o a l a n y l residue in place o f G1p 734, a n d a prolyl residue in place o f Ser-733. PFL activase f u n c t i o n e d catalytically, with s t o i c h i o m e t r i c c o n s u m p t i o n o f SAM a n d the octapep-

24

PERRYA, FREYAND SQUIREJ. BOOKER

tide with a rate constant of 1 min -1. On isolation of the peptide, and analysis by mass spectrometry and NMR, it was found to be adenosylated at the olefinic ~ carbon of the dehydroalanyl residue, indicative of addition of the 5'-deoxyadenosyl radical to the double bond, followed by quenching of the resulting C2 radical (V Wagner et al., 1999). C. Characterization of PFL-Activase

Unlike PFL, PFL-activase is distinctly a metalloenzyme. It was initially isolated in 1969, and shown to require incubation with ferrous iron and dithiols before it could convert PFL into its active form (Knappe et al., 1969). Further studies showed it to be a monomeric protein of approximately 30 kDa, with UV-visible features that were indicative of a covalently b o u n d cofactor. The optical spectrum displayed a broad absorption from 310 nm to 550 nm with a discrete peak at 388 nm, and a shoulder at 430 nm (Conradt et al., 1984). The determination of the primary structure of PFL-activase from its nucleotide sequence, which consists of 246 amino acids, established it to be a 28-kDa protein (R6del et aL, 1988). The N-terminal domain of the protein shared a significant degree of similarity with the same region of gene 55.9 of bacteriophage T4 (R6del et al., 1988; Wong et al., 1993). In particular, both proteins contained a cluster of cysteine amino acids, which conspicuously resembled a metal binding site. The bacteriophage T4 gene was located just downstream of the s u n Y intron (split gene unknown, why), which is now known to encode an anaerobic ribonucleotide reductase (Wong et al., 1993; Young et al., 1994). T4 gene 55.9 PFL-activase

FVTGCLHKCEGCYNRSTW F F Q G C L M R C L Y C H N R D YW

Initial expression of PFL-activase in E. coli u n d e r the control of a heat-inducible p r o m o t e r resulted in protein that was mainly p r o d u c e d in an insoluble form. Solubilization of the inclusion bodies, followed by gel filtration u n d e r denaturing conditions, gave protein that was >95% homogeneous. Refolding of the protein and reconstitution with Fe(II) yielded 3 to 5 mg of PFL-activase from an initial 0.8 g of cell paste (Wong et al., 1993). The specific activity of the reconstituted protein (~2 nmol rain -1 mg "q) was somewhat lower than that which had been previously obtained directly from E. coli K12 grown anaerobically on a glucose mineral medium (-25 nmol min -1) (Conradt et al., 1984). The presence of an iron-sulfur cluster in PFL-activase was first established in the laboratories of Broderick and Johnson, using several analyti-

S-ADENOSYLMETHIONINE-DEPENDENT ENZYMES

2-r')

cal and spectroscopic techniques. T h e enzyme o n which these studies were c o n d u c t e d was f r o m an overexpression system similar to that of Wong a n d co-workers (Wong et al., 1993); however, PFL-activase was purified f r o m the soluble extract u n d e r an argon a t m o s p h e r e (Broderick et al., 1997). T h e purified enzyme was red-brown in color, and its UV-~fisible spectrum was consistent with the presence of an iron-sulfur cluster, kaaalyses for iron and acid-labile sulfide revealed 1.5 + 0.1 mol of the f o r m e r and 1.7 + 0.2 mol o f the latter per mol o f enzyme m o n o m e r . T h e iron-sulfitr clusters existed as mixtures of [4Fe-4S] +2 and [2Fe-2S] +2 configurations, as d e t e r m i n e d by variable t e m p e r a t u r e magnetic circular dichroism and resonance Raman spectroscopies (Broderick et al., 1997). These diamagnetic states o f as-isolated PFL-activase were also consistent with the absence o f an EPR signal in the g = 2 region. ,an EPR-detectable signal was also not g e n e r a t e d o n incubation with dithionite. T h e c o n c o m i t a n t slight bleaching of the UV-visible spectrum indicated either partial cluster destruction, or conversion o f some o f the [2Fe-2S] +~ clusters into [4Fe-4S] +2 clusters. As in the case o f lysine 2,3-aminomutase, reduction was achieved with dithionite only in the presence o f SAM (Lieder et al., 1998). T h e d o m i n a n t resonance was mainly axial, and was well simulated with gvalues o f 2.013, 1.889, and 1.878 (Broderick et al., 1997). Characterization o f the iron-sulfur clusters o f PFL-activase was also carried out in the K n a p p e laboratory, (Kfilzer et al., 1998). T h e e n z y m e was o v e r e x p r e s s e d b e h i n d a tac p r o m o t e r in an E. coli strain lacking a functional PFL-activase, a n d purified f r o m the soluble p o r t i o n o f the cell extract. T h e purification was carried out in a m a n n e r such that the final p r o t e i n was devoid of iron, a l t h o u g h it was apparently p r o p e r l y fblded. T h e iron-sulfur cluster was subsequently reconstituted by i n c u b a t i o n u n d e r a n a e r o b i c conditions with Fe(II) a n d S ~- in the p r e s e n c e o f 100 m M 2 - m e r c a p t o e t h a n o l . Protein purified in this m a n n e r had a specific acti~4ty o f 60 n m o l rain -t m g q u n d e r standard assay conditions, which was twofold greater than what h a d b e e n o b t a i n e d f r o m E. coli K12 (Conradt et al., 1984). T h e p r o t e i n c o n t a i n e d 2.6 _+0.2 equiv, of iron, and 2.5 + 0.2 equiv, o f sulfide p e r p o l y p e p t i d e chain, and its UV-visible spectrum was indicative o f the p r e s e n c e o f [4Fe-4S] +e clusters. T h e reconstituted protein also displayed a weak, nearly isotropic signal c e n t e r e d at ,g = 2.026, indicative o f a low a m o u n t o f [4Fe-4S] +1 cluster type. In contrast to the earlier studies o f Broderick et al., the [4Fe-4S] +e clusters were readily r e d u c e d to the S = 1/2 [4Fe-4S] +l state on t r e a t m e n t with dithionite. T h e resulting spectrum was axial, displa}4ng g values of 2.029 and 1.925. Addition o f SAM or ~gadenosyl-L-homocysteine (SAH) to the EPR-active e n z y m e p e r t u r b e d the spectrum markedly, transforming the axial signal into a clear r h o m b i c signal (KSlzer et al., 1998),

26

PERRYA. FREYAND SQUIREJ. BOOKER

PFL-activase contains 6 cysteine amino acids, and each of them was changed to a serine by site-directed mutagenesis (R6del et al., 1988; K61zer et al., 1998). The corresponding proteins were purified and reconstituted as described for the wild-type protein. Only the C29S, C33S, and C36S variants afforded proteins with negligible activase activities, while the remaining variants (C12S, C94S, and C102S) were almost as active as the wild-type protein. Studies of SAM binding to each of the variant proteins were consistent with their activity profiles, since only the C12S, C94S, and C102S variants b o u n d SAM with any measurable affinity. The SAM/holoenzyme complex was stable to gel filtration u n d e r anoxic conditions, and the Kd of the complex was estimated to be ~3 ~tM. These experiments indicate that Cys-29, Cys-33, and Cys-36 provide ligands to the iron-sulfur cluster, and that the presence of the cluster is a major determinant for SAM binding to PFL-activase (K61zer et al., 1998). The cleavage of SAM to 5'-deoxyadenosine, methionine, and PFL-Ea requires an electron, which can be provided by a n u m b e r of sources, as described above. The Broderick laboratory showed that the reduced [4Fe-4S] +~ cluster of PFL-activase is a probable intermediate in the activation reaction, providing the electron necessary for SAM cleavage (Henshaw et al., 2000). In an experiment similar to that carried out with the anaerobic ribonucleotide reductase (Ollagnier et al., 1997), PFLactivase was photoreduced in the presence of 5-deazaflavin for varying lengths of time. Subsequent to the addition of SAM and the removal of illumination, which results in elimination of exogenous reductant, EPR spectra were recorded to detect formation of the [4Fe-4S] +1 cluster. A portion of each time point was also used to detect formation of the glycyl radical after addition of PFL-Ei. For each time point, the concentration of the glycyl radical closely matched the concentration of the [4Fe-4S] +l cluster (Henshaw et al., 2000). II.

ANAEROBIC RIBONUGLEOTIDE REDUCTASE

A. Molecular Properties and Reaction

Ribonucleotide reductases play a pivotal role in DNA biosynthesis by providing the cell its only means of forming 2'-deoxyribonucleotides, which is t h r o u g h the reduction of the corresponding ribonucleotide (Reichard, 1993a). T h r e e classes have been characterized in varying detail, and differ primarily in the metallocofactor that they employ in effecting the transformation. Class I enzymes, for which the "aerobic" reductase from E. coli is the prototype, employ a

5-ADENOSYLMETHIONINE-DEPENDENT ENZYMES

~7

diiron center-tyrosyl radical cofactor, while class II enzymes, for which the reductase from Lactobacillus leichmannii is the prototype, employ coenzyme B12. In these classes of reductases, the role of the cofactor is to generate a transient protein-based cysteine radical, which initiates catalysis by abstracting the 3'-hydrogen atom of the nucleotide substrate. Two excellent reviews of the ribonucleotide reductases have been written, and the reader is invited to consuh them c o n c e r n i n g details of the reaction (Jordan and Reichard, 1998: Stubbe and van der Donk, 1998).

ppp.~

I~ OH OH

ppp_...~ ¢3 ~

H+ + H/~lO O ~ ' -

I~ OH H

C O + H20

N = adenine, guanine, cytosine, uracil Biochemical studies carried out in the late 1980s provided evidence for a ribonucleotide reductase in E. coli that was distinct from the class I enzyme, and which was not coenzyme Bl2 d e p e n d e n t (Fontecave el a[., 1989; Reichard, 1993b). Extracts from a strain of E. coli unable to grow aerobically contained an oxygen sensitive activity that was able m reduce cytidine triphosphate to deoxycytidine triphosphate. This activity required SAM and a loosely b o u n d metal. Subsequent fractionation of the extract implicated a n m n b e r of proteins and factors that were required for turnover. One protein, dAB, was purified by affinity chromatography on dATP-sepharose, a procedure that is routinely used to isolate the class I reductase. This resin exploits the ability of many of these enzymes to bind dATP, which is an allosteric regulator of reductase activity (Eriksson and Sj6berg, 1989). The flow-through also contained at least one essential protein, dA1, which was purified by conventional chromatographic techniques. Other factors that were crucial for activity were stable to boiling for 30 min. One of these factors, K+, bound tightly to Chelex resin, and was eluted only in 12 M HCI. Two remaining factors were not retained by the Chelex column, and were later identified as formate (Mulliez el cd., 1995) and flavodoxin (Bianchi el aL, 1993a). In contrast to class I and class II reductases, which obtain their reducing equivalents from NADPH via the thiored o x i n / t h i o r e d o x i n reductase reducing system, the anaerobic reductase

28

PERRY A. FREY AND SQUIRE J. BOOKER

obtains its reducing equivalents via the oxidation of formate to C O 2 (Mulliez et al., 1995). Intriguingly, formate is a product of the reaction catalyzed by pyruvate formate-lyase (detailed above), another key enzyme in the anaerobic life cycle of E. coli. Isolation and characterization of fraction dA1 showed it to be a single polypeptide of ~28 kDa, and it was provisionally termed "activase" because of its similarity in size with the PFL-activase and its requirement for turnover (Eliasson et al., 1993). Although it was clear from N-terminal sequence analysis that this protein was not PFL-activase, it was only after its amino acid sequence was determined that its identity was revealed to be ferredoxin (flavodoxin) NADP + reductase (Bianchi et al., 1993b). The cloning and sequencing of the nrdD gene, which encodes the anaerobic reductase (ARR), provided evidence for a close relationship between it and pyruvate formate-lyase. The enzymes share stretches of sequence similarity, which are particularly noteworthy at their C termini. In addition, ARR shares m o r e than 72% sequence identity with the bacteriophage T4 s u n Y g e n e , which was of unknown function at the time. Of particular interest was the sequence T R R V C G Y L (bold letters indicate identical residues), which was highly similar to the sequence in PFL that was known to contain the glycyl radical (T I R V S G YA) (Sun et al., 1993). Site-directed mutagenesis and isotopic labeling studies confirmed the presence of a glycyl radical in the anaerobic reductase. After purification and activation of the enzyme, it displayed an isotropic EPR signal centered at g= 2.0033, having a doublet splitting of 14 to 15 G (Mulliez et al., 1993; Sun et al., 1996). In the presence of [2H]glycine-containing enzyme, the doublet splitting was largely absent, demonstrating that it originated from hyperfine coupling of the radical to the hydrogen of glycine. The [2-13C]glycine-containing enzyme displayed a m u c h broader and less resolved signal, providing evidence that a large degree of unpaired spin resided on carbon 2 of glycine. Substitution of glycine 681 with alanine abolished the EPR signal as well as catalytic activity. Exposure of the radical-containing enzyme to oxygen resulted in C-terminal truncation of the protein at the glycyl radical site, producing 77and 74-kDa fragments (Sun et al., 1996). There are distinct differences between the glycyl radical spectrum of PFL and ARR. Unlike the glycyl radical of PFL, the or-hydrogen of the ARR glycyl radical does not exchange with solvent, even over a period of 24 hours. In addition, the spectrum of the PFL radical is more complex. It possesses partially resolved sub-doublet splitting, arising from two nonexchangeable protons (Wagner et al., 1992; Sun et al., 1996) (Figure 10).

.5 mT •

i

A

B

C

_J !

I

!

0

2

4

5= mT

/

Fit_;. 10. X-band EPR spectra of the glycyl radicals of ARR-reductase a n d PFL-activase. Spectrum A is that of activated pyruvate formate-lyase taken in | H 2 0 . Spectrum B is that of the same protein after exchange into 2H20. Both spectra were obtained at 253°K with a modulation amplitude of 1.4 G. Spectrum C is that of the anaerobic ribonucleotide reductase in IH20. It was recorded at 100°Kwith a modulation amplitude of 1.6 G. The spectrum does n o t change significantly after exchange of the protein into 2H20. The spectra are r e p r o d u c e d from Knappe and Volker Wagner (1995) a n d Ollagnier et al. (1996) and used with permission from the American Society for Biochemistry and Molecular BioloD,, Inc., a n d Academic Press, Inc. 29

30

PERRY A. FREY AND SQUIRE J. BOOKER

B. Characterization of the Activase Subunit and the Role of SAM

Early isolation o f ARR led to its assignment as a h o m o d i m e r i c protein (0~2) of 154 kDa, which was capable of catalyzing its own activation in the presence of SAM, KC1, DTT, and an electron source (Eliasson et al., 1992; Sun et al., 1993). As in PFL-activase, the electron source can be the flavodoxin/flavodoxin reductase reducing system, dithionite, or 5'-deazaflavin plus light (Mulliez et al., 1993). The near h o m o g e n e o u s preparations contained iron and sulfide in a 1:1 stoichiometry (0.5 to 1.3 F e / d i m e r ) , and displayed EPR signals (N0.12 spins/total iron) that were indicative of an [3Fe-4S] +1 cluster (Mulliez et al., 1993). The s u b s e q u e n t finding of an o p e n reading frame with s e q u e n c e similarity to PFL-activase immediately downstream of nrdD led to a re-examination of previous experiments (Sun et al., 1995). O n heavily overloaded SDS-PAGE gels, a faint band was visible, which migrated as a 17-kDa polypeptide, and which was estim a t e d to comprise no m o r e than 1% of the total protein. Its subseq u e n t isolation showed it to be brown in color, and to display a UV-visible spectrum indicative of an iron-sulfur cluster. The 17.5-kDa protein (as determined from its nucleotide sequence) was overexpressed in a T7-based system, purified, and then characterized biochemically and spectroscopically. The protein eluted from a Superdex-75 column (in the absence of DTT) in two fractions, corresponding to monomeric and dimeric subunit structures. The dimeric fraction contained 0.1 to 0.25 Fe/polypeptide chain and was colorless, while the monomeric fraction was essentially void of color and contained only 0.01 to 0.02 irons/polypeptide chain. The protein during chromatography in the presence of DTT retained larger amounts of iron. Either fraction could be reconstituted with iron and sulfide u n d e r anaerobic conditions to give a total of 2 equiv, of each per polypeptide, with the monomeric fraction subsequently eluting as a mixture of monomer, dimer, and more aggregated states. Using 59Fe, it was found that the iron was tightly b o u n d in the dimeric state, but was lost upon dissociation to the monomeric state (Ollagnier et al., 1996). The authors suggested that the cluster might be b o u n d at the interface of two subunits, with cysteine ligands being contributed by each (Ollagnier et al., 1996). Very recently, the notion of a subunit bridging cluster has become less attractive, since the same laboratory has shown by M6ssbauer and EPR studies that each polypeptide can accommodate one [4Fe-4S] cluster when apoprotein is reconstituted u n d e r strictly anaerobic conditions (Tamarit et al., 1999). The reconstituted protein displayed a weak EPR signal centered at g -- 2.3, indicative of an [3Fe--4S] +1 cluster. U p o n incubation with dithion-

S-ADENOSYLMETHIONINE-DEPENDENT

ENZYMES

~;~1

ite, this signal disappeared, and was replaced by a rhombic signal with features at g = 2.02, 1.92, and 1.88. Temperature and power dependence studies of the signal were suggestive of an [4Fe-4S] +1 cluster, and not an [2Fe-2S] +1 cluster (Ollagnier et al., 1996). In the presence of air, the [4Fe-4S] clusters are converted back to [2Fe-2S] clusters, and can be reconverted to [4Fe-4S] clusters u n d e r strongly reducing conditions (Tamarit et al., 1999). Centrifugation in sucrose density gradients established an 0t2~2 structure of the anaerobic ribonucleotide reductase, in which the 0t2 subunit shifts the equilibrium of the [~ subunit to the ditneric form, and the resulting 0t2 and [~2 subunits associate tightly in a 1:1 complex (Ollagnier et al., 1996). However, the association is not absolute, because the ~2 c o m p o n e n t of the complex has recently been shown to be capable of activating multiple 0t2 components. Therefore, as in the case of PFL, it appears to act as an activase rather than a component of an 0t2[~2 holoenzyme (Tamarit et al., 1999). All known radical SAM enzymes contain a C X X X C X X C motif (see sequence a l i g n m e n t in next section), which is generally believed to contain the cysteines that ligate the iron-snlfur cluster. As described above, this has been shown to be the case for PFL-activase. Alteration of these cysteines in the ~ protein (17.5 kDa protein) of ARR p r o d u c e d similar results (Tamarit el al., 1999). The protein contains 5 cysteines, and each was c h a n g e d to alanine, purified, and reconstituted u n d e r conditions that were similar to the wild-type protein. Two variants (C19A and C96A) behaved essentially as the wildtype protein with respect to their activities and spectroscopic properties. The remaining variants (C26A, C30A, and C33A), that comprised the C X X X C X X C motif, were inactive. Interestingly, each of these variants was able to a c c o m m o d a t e iron and sulfide almost as well as the wild-type protein, and their UV-visible spectra indicated that they contained [4Fe-4S] clusters. One variant, C30A, was analyzed by M6ssbauer spectroscopy. Approximately 82% of 1he total iron was in [4Fe-4S] +~ or [3Fe-4S] II configurations, of which 54% was attributed to the [4Fe-4S] +~ state (Tamarit et al., 1999). This would suggest that [4Fe-4S] +e clusters could assemble with onlv two cysteine ligands. However, the authors point out that it is possible that Cys-19 or Cys-96 could have been recruited for stabilizing the clnster, as could have DTT. The anaerobic ribonucleotide reductase was the first of the radicalSAM enzymes in which the reduced iron-sulfur cluster was shown to supply the electron necessary for SAM cleavage (Ollagnier et al., 1997). Analogously to PFL-activase, protein ~ of the ARR is easily reduced to

32

PERRY A. FREY AND SQUIRE J. BOOKER

the [4Fe-4S] +1 state on addition of dithionite or deazaflavin plus light. The Fontecave laboratory exploited the characteristic of the deazaflavin reducing system wherein reducing equivalents are eliminated when illumination is halted (Ollagnier et al., 1997). In the presence of the flavodoxin reducing system or 5-deazaflavin plus light, the cz2~2 complex of ARR catalyzed the reduction of SAM to methionine (which was quantified), and presumably 5' -deoxyadenosine (which was not quantified). This reaction, which the ~2 subunit alone could catalyze at a reduced rate, was greatly stimulated by DTT (8 mM). The reaction could be dissected into two distinct steps by photoreduction in the absence of SAM. U p o n removal of illumination of the [~2 subunit only, SAM was added u n d e r conditions of no light, and changes in the EPR spectrum of the [4Fe-4S] +1 cluster as well as the production of methionine were monitored as a function of time. These two processes took place approximately on the same time scale. In an analogous experiment in the presence of the cz2~2 complex, the glycyl radical formed at a rate that was similar to methionine production (Ollagnier et al., 1997). V. BIOTIN SVNTHASE

A. Biotin Synthase Reaction and Molecular Properties The synthesis of biotin from its direct precursor, dethiobiotin, is an intriguing transformation that entails the insertion of a sulfur atom between two unactivated carbons (C-1 and C-4) of the precursor. Despite efforts extending over 20 years, the reaction mechanism remains largely unresolved. In fact, much of what is known about this step in biotin synthesis has been obtained from in vivo feeding studies conducted primarily in the laboratories of Parry and Marquet. A review of these early studies on biotin synthase, as well as lipoic acid biosynthesis, has previously been published, and is an excellent source of details on these experiments (Parry, 1983). Parry's laboratory synthesized dethiobiotin specifically labeled with tritium at C-1, C-2, C-3, or C-4, and then administered each radioactive c o m p o u n d to a growing culture of Aspergillus niger in combination with [10-14C] (+)-dethiobiotin as a dual label. Subsequent to isolation and purification of the corresponding biotin sulfone methyl esters, their tritium to C-14 ratios were determined. The results were consistent with removal of only 2 hydrogens from dethiobiotin in its conversion to biotin. O n e hydrogen is removed from C-l, while the other is removed from C-4. Removal of tritium from C-4 was stereoselective, with only the

,~-ADENOSYLMETHIONINE-DEPENDENT ENZYMES

2~.~

4-pro S isomer of dethiobiotin giving rise to significant tritium loss (Parry and Kunitani, 1979; Trainor et al., 1980). Since the absolute

stereochemistry of biotin at C-4 is known to be S, this indicates that sulfur is introduced at this position with retention of configuration. Similar results in E. coli were obtained by Marquet's laboratory using deuterated precursors and analysis by mass spectrometry (Frappier et al., 1982). Derivatives of dethiobiotin that were hydroxylated at C-1 or C-4 were not converted to biotin by A. niger;" however, resting cells of B. sphaericus converted the C-1 thiol derivative of dethiobiotin to biotin without loss of 3~S label (Frappier et al., 1979; Marquet et al., 1993). O

o

HN' H

HNkNH H

H

s 1 -

V

V

~COOH

Dethiobiotin

OOH Biotin

The nucleotide sequence of the bio operon of E. coli was determined in 1988, and conversion of dethiobiotin to biotin in cell-free extracts of E. coliwas demonstrated in 1992 (Ifuku et al., 1992; Otsuka et al., 1988). In this study, a recombinant plasmid containing the bioB gene cloned behind a tac p r o m o t e r was the source of the enzyme, and biotin synthesis was detected by a microbiological assay. Enzyme activity required fructose 1,6-bisphosphate, Fe (II), SAM, NADPH, KC1, and dethiobiotin (Ifuku et al., 1992). A m m o n i u m sulfate fractionation of the cell-free extract abolished activity; however, it could be reconstituted upon recombining the fractions. It was therefore concluded that the bioB gene product supports biotin formation only in concert with 1 or more other proteins (Ifuku et al., 1992). Parallel work in another laboratory showed that biotin biosynthesis required the flavodoxin/flavodoxin reductase reducing system and a thiamine pyrophosphate-dependent protein (Birch et al., 1995). This activity was d e p e n d e n t on several low molecular weight compounds, including SAM, cysteine, thiamine pyrophosphate, Fe (II), NADPH, and either asparagine, aspartate, glutamine, or serine. Experiments were carried out with cell-free extracts containing an overexpressed E. coli bioB gene product; however, TLC and autoradiography, or HPLC with online radiochemical detection, was used to quantify biotin formation. Using this assay, 35S was found to be incorporated into biotin when the cell-free extract contained -35S-labeled cysteine (Birch et al., 1995). This result corroborated earlier in vivo labeling studies with 34802- 4 and

34

PERRY A. FREY AND SQUIREJ. BOOKER

L-[sulfane-348] thiocystine, in which cysteine was implicated as providing the sulfur atom for biotin synthesis (DeMoll et al., 1984). Similar studies were also carried out in Marquet's group using the bioB gene from B. sphaericus, and further established its relatedness with PFL-activase and ARR activase (M6jean et al., 1995). Using highly purified bioBprotein (80% pure, 1 mg m1-1) in the presence ofB. sphaericus cell-free extract (10 mg ml-l), or with 5-deazaflavin and light replacing the cell-free extract, biotin was shown to be p r o d u c e d from dethiobiotin in the presence of only SAM, cysteine, and DTT. Interestingly, in assays containing cell-free extract, ~5S from 35S-labeled cysteine was incorporated into biotin. However, in assays in which 5-deazaflavin plus light replaced the cell-free extract, 35S from 35S-labeled cysteine was not incorporated into biotin (M6jean et al., 1995). The stoichiometry of biotin formation with respect to SAM consumption has been established, and defines a new class of enzymes within the radical-SAM family, which are distinct from those that employ SAM as a cofactor, or those that use it to generate a cofactor. Using radioactive forms of SAM, Marquet's laboratory found that it was cleaved to methionine and 5'-deoxyadenosine in a 1:1 ratio u n d e r conditions necessary for biotin formation (Guianvarc'h et al., 1997). Careful quantitation showed that the ratio of either of these products to biotin formed was approximately 3:1. Similar results were obtained with the bioB gene product from both E. coli and B. sphaericus, and in the presence of 5deazaflavin or the flavodoxin/flavodoxin reductase reducing system. It was concluded from this work that biotin biosynthesis most likely proceeds with consumption of 2 equiv, of SAM per equiv, of biotin (Scheme 3), and that the third equiv, observed is probably from abortive processes (Guianvarc'h et aL, 1997). The stoichiometry of SAM consumption was corroborated by Shaw's group (Shaw et al., 1998). Using HPLC with online radiochemical monitoring, they detected an enzyme-generated intermediate in biotin synthesis. The intermediate was derived from [aaC]dethiobiotin, and also contained 35S from [35S]cystine. The intermediate was distinctly separated from biotin or dethiobiotin, and required SAM for its production. Most excitingly, it could be isolated from the reaction mixture by chromatography, reintroduced into another assay, and shown to be converted into biotin. However, this step also required SAM. Subseq u e n t careful quantification of the intermediate, methionine, and biotin as a function of time, established that 1 equiv, of SAM is required for synthesizing the intermediate from dethiobiotin, and another equiv, is required for synthesizing biotin from the intermediate (Shaw et al., 1998).

.++ ~+

5 +-'+

o%~

,'<

~P+.,

+"

~ II

o

,-

~5

"~

+~

36

PERRYA. FREYAND SQUIREJ. BOOKER

B. Characterization of the Iron-Sulfur Clusters of the bioB Protein Isolation and spectroscopic characterization of the proteins e n c o d e d by the bioB genes of E. coli and B. sphaericus revealed that they contained iron-sulfur clusters (M6jean et al., 1995; Sanyal et al., 1994). The protein from B. sphaericus contained 0.7 mol of iron per mol of m o n o m e r (M6jean et al., 1995), whereas the protein from E. coli contained 1.7 to 2.1 mol of iron and 2.0 mol of acid-labile sulfide per mol of m o n o m e r (Sanyal et al., 1994). The E. coli protein displayed a strong peak at 330 rim, a strong peak at 453 nm, a weaker feature at 420 nm, and a shoulder at 540 nm, collectively indicative of a [2Fe-2S] cluster. These same features could be seen in the UV-visible spectrum of the B. sphaericus protein, although they were less distinct. The as-isolated E. coli protein was EPR silent, and subsequent reduction with dithionite did not readily produce a signal. However, reduction with dithionite over an extended period of time afforded a weak rhombic spectrum with gavg = 1.95, and distinct features at g= 2.03 and g = 1.90 (Sanyal et al., 1994). The iron-sulfur centers of the bioB protein from E. coli have been characterized by a n u m b e r of spectroscopic methods. Variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman spectroscopies confirmed that the protein in its aerobically purified state contains diamagnetic S = 0 [2Fe-2S] 2+ clusters. On anaerobic reduction with dithionite in the presence of 60% ethylene glycol or glycerol, a near-stoichiometric conversion of the [2Fe-2S] 2+ clusters to [4Fe-4S] 2+ clusters occurs, even in the absence of added iron or sulfide. The Raman Fe-S stretching frequencies indicated that the [2Fe-2S]2+ clusters were incompletely coordinated by cysteines; however, the resulting [4Fe-4S] 2+ clusters were completely cysteinyl-S coordinated. The E. coli enzyme has a molecular weight Mr = 39,000, and runs as a dimer on native gels. It was suggested, therefore, in light of the stoichiometry of cluster conversion, that each m o n o m e r of biotin synthase contains an [2Fe-2S] 2+ cluster that can reductively dimerize to form [4Fe-4S] 2+ clusters at the interface of two subunits (Duin et al., 1997). Treatment of aerobically purified biotin synthase with dithionite for long periods of time, followed by addition of glycerol or ethylene glycol, resulted in generation of an [4Fe-4S] 1+ cluster (>0.5 spins per homodimer), as determined by EPR spectroscopy and VTMCD. The spectrum was suggestive of a mixed spin system (S -- 1/2 and S --- 3/2), and displayed gvalues of 2.044, 1.944, and 1.914 for the S = 1/2 state (Duin et al., 1997). The [2Fe-2S] z+, [4Fe-4S] 2+, and [4Fe-4S] l+ cluster forms have also been confirmed by M6ssbauer spectroscopy, as have their ability to be interconverted (Ollagnier-De Choudens et al., 2000; Tse Sum Bui et al.,

S-ADENOSYLMETHIONINE-DE PENDENT ENZYMES

37

1999). These studies were carried out on reconstitued biotin synthase, rather than enzyme possessing the iron and sulfide content that accompanies purification by the normal procedure. By performing the reconstitution with starting protein that was devoid of iron-sulfur clusters, and by adhering to strict anaerobicity, one group was able to incorporate 1 [4Fe-4S] cluster per monomer. They concluded therefore that the [4Fe-4S] clusters were probably not bridging two subunits (Ollagnier-De Choudens et al., 2000). Recently, Jarrett's group has shown that conversion of the [2Fe-2S] cluster form of biotin synthase to the [4Fe-4S] cluster form involves dissociation and reassociation of iron, rather than reductive dimerization (Ugulava et aI., 2000). The bioB protein possesses the iron-sulfur cluster-binding motif that is c o m m o n to all radical-SAM enzymes, and the role of this motif in biotin synthase has been studied experimentally. Each of the participating cysteines in the binding motif has been altered to alanine, and the corresponding protein has been purified and characterized spectroscopically and biochemically. Each of the variant proteins were inactive with respect to biotin synthesis; however, they exhibited UV-visible spectra that were similar to the wild-type protein. Their iron content was approximately half of that of the wild-type protein, and their spectral extinction coefficients between 300 and 800 nm were lower. In addition, their circular dichroism spectra did not suggest gross secondarv structure changes. From these results, it was concluded that cysteines 53, 57, and 60 most likely contribute ligands to the iron-sulfur cluster of biotin synthase (Hewitson et al., 2000). Experiments to probe the nature of the fourth ligand of the [FeS] cluster of biotin synthase have not been reported. As is the case in PFL-activase and ARR-activase, it is unlikely to be another cysteine on the protein.

E. E. E. E. C

lron sulfur cluster binding motifs for coli biotin synthase coli lipoic acid synthase coli PFL-activase coli A R R activase subterminale 2,3-aminomutase

radical-SAM enzymes A C P E D C KY C P ICTRRCPFCD G C L M R C LYC H G CV H E C P G CY M C SMYC R H C T

C. Nature of the Sulfur Donor The ability to generate apobiotin synthase and reconstitute it facilitated experiments to investigate the nature of the sulfur d o n o r in the reaction. Upon reconstituting the [4Fe-4S] cluster of either the E. coli bioB apoprotein or the B. sphaericus apoprotein with iron and Na2 [348], 348 was f o u n d to be transferred to biotin in standard assays

38

PERRYA. FREYANDSQUIREJ. BOOKER

using 5-deazaflavin and light or the flavodoxin/flavodoxin reductase reducing system (Bui et al., 1998). The incorporation was approximately 65% o f the starting s4S content, indicating that partial exchange of the cluster-bound sulfur with some other source took place (Bui et al., 1998). Similar results were found in another study on the origin of the sulfur d o n o r using protein isolated from cultures grown in the presence of [35S] methionine, [35S]cysteine, a n d / o r [35S]sulfide. With protein from cultures grown in the presence of [35S]methionine only, almost no radioactivity was found in isolated biotin, while in samples containing protein isolated from cultures containing both [35S]methionine and [35S] cysteine, a meager a m o u n t of radioactivity was present. In contrast, when protein isolated from cultures grown on [35S]sulfide and [35S]cysteine was used, significant transfer of radioactivity to biotin was obtained (Gibson et al., 1999). These experiments are consistent with the sulfur d o n o r in the biotin synthase reaction as the iron-sulfur cluster of biotin synthase itself. No group has yet been able to demonstrate m o r e than one turnover in assays of biotin synthase. In the fully reconstituted protein, one full turnover was obtained, while in the protein containing 2 equiv, of Fe per m o n o m e r , 0.5 turnovers were obtained (OllagnierDe Choudens et al., 2000). The observation that the sulfur in biotin is derived from the [FeS] cluster would suggest that assay c o m p o n e n t s that function to reconstitute the cluster might r e n d e r the reaction catalytic. The Marquet laboratory d e m o n s t r a t e d that the NifS protein as well as rhodanese were effective in mobilizing sulfur from cysteine for iron-sulfur cluster reconstitution into apobiotin synthase; however, they had no effect on turnover in the in vitro assay (Tse Sum Bui et al., 2000).

VI. LIPOlC ACID BIOSYNTHESIS

The biosynthesis of lipoic acid in E. coli bears remarkable similarities to the biosynthesis of biotin. Early in vivo feeding studies by Reed showed that octanoic acid was the direct precursor oflipoic acid (Parry, 1983). These studies were later corroborated by Parry, in which he showed that [1-14C]octanoic acid was specifically incorporated into lipoic acid when administered to shake cultures of the bacterium (Parry, 1983); Parry and Trainor, 1978). When [5-3H] -, [6-all], [7-3H], or [8-3H]octanoic acid was used, only the molecule labeled at C-6 showed substantial tritium loss, suggesting that lipoic acid biosynthesis proceeds without intermediate desaturation (Parry, 1977, 1983).

S-ADENOSYLMETHION1NE-DEPENDENT ENZYMES

8

6

4

814 SHH

2

octanoic acid

dihydroiipoic acid

1

.~0

2

S~S 4

H

lipoic acid

The stereochemistry of sulfur introduction at C_,-6was investigated using octanoic acid that was stereospecifically tritiated at that position. [ (6S3-63HI- and [(dR)-6-SH]octanoic acid were synthesized, and separately administered to growing cultures of E. coli after mixing with a defined amount of [ 1J4C] octanoic acid. Subsequent to derivatizing the lipoic acid that was isolated, it was found that only [ (dR)-6--~H]octanoic acid gave rise to significant tritium loss (Parry, 1983; Parry and Trainor, 1978). This indicated that the insertion of sulfur at C-6 proceeds with inversion of configuration, in clear contrast with the biosynthesis of biotin from dethiobiotin, which proceeds by retention at the analogous carbon (Parry, 1983). Hydroxylated intermediates at C-6 a n d / o r (;-8 were ruled out based on the inability" of either [6(RS)-2H1]-6-hydroxyoctanoic acid, [8-9H~]-8 hydroxyoctanoic acid, or [8-2H2]-(+)-6,8-dihydroxyoctanoic acid to be converted into lipoic acid (Parry, 1983). Howevm; when [8-eHe]-8-thiooc tanoic acid was administered to E. coli, a substantial percentage of the isolated lipoic acid was derived from it. [6(RS)-2Hl]-6-thiooctanoic acid was significantly worse as a precursor (Parry, 1983). Genetic and biochemical studies hinted that both sulfur insertions into octanoic acid were d e p e n d e n t on the activity of one central protein, the product of the lipA locus (Hayden et al., 1993; Herbert and Guest, 1968; Van den Boom et al., 1991). The nucleotide and axnino acid sequences for the E. coli lipA gene were established by two groups in the early 1990s (Hayden et al., 1992; Reed and Cronan, 1993). The protein contains 321 amino acids, and has a calculated molecular weight of 36,061. It also has substantial sequence similarity with the bioB protein of E. coli. Initial overexpression of the protein resulted in its production in inclusion bodies, from which it was readily purified b} solubilization with guanidine hydrochloride and gradual dialysis against a renaturation buffer (Reed and Cronan, 1993).

lipA bioB

Sequence similarities between bioB and lipA proteins CTRRCPFC lipA DVFNHNLENVPRIY CPEDCKYC bioB DYYNHNLDTSPEFY

lipA bioB

LERFKEA LEKVRDA

lipA bioB

SGLMVGLGET SGGIVGLGET

lipA DEFLEMKAEAIA bioB DAFDFIRTIAVA

Further studies in the laboratories of Marietta and Fontecave established that the lipA gene product is an iron-sulfur protein (Busby et al.,

40

PERRYA. FREYAND SQUIRE J. BOOKER

1999; Ollagnier-de Choudens and Fontecave, 1999). The protein existed in both m o n o m e r i c and dimeric states, and as isolated it contained 1.8 _+ 0.2 tool of Fe, and 2.2 -+ 0.4 tool of acid-labile sulfide per tool of monomer. The presence of [2Fe-2S] and [4Fe-4S] clusters was determined from resonance Raman and UV-visible absorption spectra (Busby et al., 1999). The protein was also purified from solubilized inclusion bodies followed by gel-filtration chromatography, and has been reconstituted with 1 [4Fe-4S] cluster per monomer, as in the case of biotin synthase (Ollagnier-De Choudens and Fontecave, 1999; Ollagnier-De Choudens et al., 2000). The reconstituted protein displayed a relatively weak EPR signal, indicative of a [4Fe--4S] +1 cluster, when reduced with 5-deazaflavin plus light (Ollagnier-De Choudens and Fontecave, 1999; OUagnier-De Choudens et al., 2000). These cluster forms have recently been confirmed by M6ssbauer spectroscopy (Ollagnier-De Choudens et al., 2000). Not even one full turnover was demonstrated in initial studies. Insightful studies from the Cronan laboratory have implicated the acyl carrier protein in the biosynthesis of lipoic acid, and have linked lipoic acid biosynthesis to fatty acid biosynthesis. E. coli use two mechanisms for incorporation of lipoic acid into the major lipoylated proteins, the pyruvate and 0t-ketoglutarate dehydrogenase complexes. Lipoic acid from exogenous sources is delivered to these complexes by a lipoate ligase (LplA). In this reaction, ATP is used to activate the carboxyl group of lipoate prior to lipoylation of the complex. Endogenously synthesized lipoic acid is transferred to these complexes by a lipoyl transferase (LplB), which uses lipoyl-acyl carrier protein as the lipoyl d o n o r (Scheme 4) (Jordan and Cronan, 1997). The lipoate transferase also acts on octanoyl-acyl carrier protein. An elegant assay was devised to test the foregoing hypothesis, in which LipB was used to transfer any lipoyl-ACP synthesized by LipA to apo-pyruvate dehydrogenase complex (apo-PDC) (Miller et aL, 2000). The assay allowed for very minute quantities of product to be detected spectrophotometrically through observation of the catalytic reduction of 3-acetylpyridine adenine dinucleotide--an NAD analog--by the action of PDC. This would take place only on transfer of the lipoyl cofactor synthesized by LipA to apo-PDC. Therefore, a substantial amplification of the reaction takes place. Using a C-terminal histidinetagged form of the LipA protein, the authors were able to detect 0.032 mol of lipoyl-ACP synthesized per mol of LipA polypeptide. This was an important result, because turnover d e p e n d e d on LipA, octanoyl-ACP, a reductant (sodium dithionite), and LipB. The requirement for SAM was not absolute; however, SAM e n h a n c e d turnover by 10-fold, suggest-

S-ADENOSYLMETHIONINE-DEPENDENTENZYMES

41

SqS O Cytoplasmicmembrane ATP

PF~ S~S

PathwayA O

O~{/ S--S

H2N_CH2..pDC

Lp, ~ A M P O LipB ~ ' ~ H //~

PathwayB

= O

2. reduCtant 3. Sulfursource?

S-ACP

\ S--ACP O

Fattyacidbiosynthesis Cytoplasmicmembrane SCtlEME 4. Redundant pathways for incorporation of lipoate into apo-pyruvate dehvdrogenase complex. Incorporation of exogenous lipoate tbllows pathway A. A lipoate ligase activates the carhoxyl group of lipoic acid in an ATP-dependent reaction fiw subsequent transfer to the acceptor protein. Endogenous transfer of lipoate to the apopyruvate dehydrogenase complex follows pathway B. LipoyI-ACP is synthesized from octanoyl-ACP by LipA. LipB catalyzes subsequent transfer of the lipoyl group flom lipoyl-ACP to the acceptor protein.

ing that the enzyme is isolated with small amounts of SAM or a SAMderived intermediate already bound (Miller et al., 2000). Evidence suggests that the FeS cluster/SAM strategy represents the earliest strategy for generating carbon-centered radicals as intermediates in enzymatic catalysis. Because of the lability of these enzymes and their typical involvement in anaerobic metabolism, this radical-generating system is among the most recent to be characterized. Early work on 2,3-aminomutase, PFL-activase, anaerobic ribonucleotide reductase, and other FeS-containing enzymes that are not within the radical/SAM

42

PERRYA. FREYAND SQUIREJ. BOOKER

family has been instrumental in establishing suitable conditions and methods for manipulating these labile proteins. Intensive studies are underway in numerous laboratories to unravel the mechanism of radical generation, as well as to characterize possible intermediates in reactions in which the FeS cluster acts as a sulfur donor.

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