Purification and characterization of coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain ΔH

Biochimica et Bioph)'sica Acta. 1073(1991) 77-I~4

~, 1991 ElsevierScience PublishersB.V. (Biomedic~tlDtv~l~,n)0304-4165/91/~03 5o ADONIS 030441659100~51C

Purification and characterization of coenzyme F~20-dependent 5,10-methylenetetrahydromethanopterin dehydrogenate from Methanobacterium thermoautotrophicum strain AH B . W . te B r i 3 m m e l s t r o e t , C h a r l e s M . H . H e n s g e n s , J a n T. K e l t j e n s , C h r i s v a n d e r D r i f t a n d G o d f r i e d D. V o g e l s Department of Microhtolo~,'. Factdtv of Sclenct,. {,'ntler~t~ of Nuntet~t.n. Vtimegen (The Netherlands) (Received 2 May 1q°91

Key words: Methylenetetrahydromethanopterint ehydrogena.,:: 5.O,7.8-Tetrahydromethanopteon;CoenzymcF.~; Deazaflavin; Methanogene~is;( M thermoaulolrophwum ) S,10-Methylenetetrahydromethanoptelin dehydrogenase from Methanobacterium thermoautolrophicum strain d H was purified to homogeneity ~ith nearly complete recovery. The aerobically stable monofunetional enzyme catalyzed the reversible oxidation of 5,lf~-methylene-5,6,7,8-tetr',,bydromethanopterin to its 5,10-methenyl derivative. For the reaction a midpoint potential E~ = - 3 6 2 mV was calculated at 6 0 ° C . The methanogenic electron carrier coenzyme F4m was strictly required as the co-substrate. The debydrogenase (Mr 216000) was purified as an apparent hexamer o4[ six identical 36 kDa subtmit~ Oxidation of 5,10-metbylenetetrabydromethanopterin ~ to eoenzyme F ~ reduction catal~z~l by the debydroger~,se with a tunmver ,,u..b~r ~ ~ 0 s - t proceeded via a ternary complex mechanism. High coneeatrations of monovalem cstions markedly s~m',da~-d ~ re*_cfion, Introduction The methanogenic bacteria represent a group of specialists that derive their ener~,y for growth from ',he conversion of a limited number of one-carbon compounds or acetate to methane .I1,2]. Most species including the organism studied here, Methanobactermm thermoautotrophicum, are capable of performing the eightelectron reduction of CO 2 to methane. In this process a series of novel coenzymes are involved as one-carbon and electron carriers. During the conversion of the one-carbon unit at the formyl through the methyl state of reduction 5,6,7,8-tetrahydromethanopterin (H4MPT) serves as the one-carbon carrier 13-61. The compound accepts a formyl group bound to methanofuran and the Abbrcviatio;~s: H4MPT. 5,6,7,8-te~ahydromethanopterin:H~folate. 5,6.7.8-tetrahydrofolate; coenzyme F420.7,8-didemethyl-8-hydroxy-5deazariboflavin-$'-phospho~llactylslutamate; F0, 7,8-didemethyl-8hydroxy-5'-d~azaribofiavin; F*, 7,8-didewethyl-8-hydroxy-5deazariboflavin-5"-phosphate; c,¢cnzyme F42oH2, 1,5-dihydro-coen~me F420; F3~o, 8-OH adenylylated coenzyme F.u0; CHAPS. 3-[(3cholamidopropyl)dimethylammoniol- l -propanesulfonate; SDS. sodium dodecyl sulfate; HPLC, hi[h-performance liquid chromatography. Correspondence: J.T. Keltjens, Department of Microbiology.Faculty of Science. University of Nijme~-n..~'L-6225 El), Ni megen. The Netherlands.

resulting product, S-fort lyI-H4MPT is subsequently dehydrated to yield 5,10-methenyI-HaMPT [7-9], The latter is converted in two subsequent reduction reactions (!) and (2) via 5,10-methylene-H4MPT to 5methyl-H4MPT. 5.lO-methcuyI-H.,MPT + cocnzymeFa.oH; 5,10-methylene-H4MPT+ ccenzymeF.,.-.o+ H +

(1)

5.10-meth~lene-H.tMPT+ coenzymeF..~H2 5-methyi-H.tMPT+ coenzymeF4,o

(2)

in the reversible reactions (1) and (2) the deazaflavin coenzyme F4z0 acts as an electron carrier [6,10-121. 5,10-Methylene-HiMPT reductase that catalyzes reaction (2) was recently purified by us from 3,1. thermoauwtrophicum (strain AH) and characterized to some detail [12]. The enzyme that reduces 5,10-mcthenylHaMPT, 5,10-methylene-H4MPT dehydrogenase, has also been purified from the organism though only in modest yield and with low specific activity [10]. Improved purification procedures have been described [11,13] for the dehydrogenase from M. thermoauwtrophicum strain Marburg, an organism which is only distantly related to the a H strain [la I. Remarkably, coenzyme F4zodependence of the latter protein was only found in the coarse of the purification procedure [11] or

78 after exposure of the enzyme to aerobic conditions [13]. For the highiy-purified enzyme prepared under anoxic conditions protons and hydrogen, rather than coenzyme F42,, were reported to be the substrates providing the protein a hydrogenase activity [13]. In this report v,c describe a relatively simple procedure to obtain with nearly complete recovery and with high specific activity the 5,10-methylene-lt,MPT dehyd,'ogenase from M. thermoautotrophicum strain AH The aerobically purified enzyme strictly used coenzyme k.~21~ ~,s the co-substrate. In addition, the enzyme has hcen characterized to some extent with respect to subunit composition, substrate specificity and kinetic properties. Parts of the study have been published in abstract-form (Forum Mikrobiologie 12 (1989) p. 55). Materials and Methods

Preparation of cell-free extracts. M. thermoautotrophicum (strain AH, DSM 1053) was grown under hydrogen and CO 2 on a mineral medium 115] and harvested at the end of the exponential growth as described previously [16]. Cell-free extracts were prepared anaerobically at 4°C by suspending whole cells (1 : 1, w/v) in 20 mM potassium phosphate buffer (pH 6.0). The suspension was passed twice through a French Pressure cell operating at 138 MPa. Cell debris were removed by centrifugation at 18000 x g for 20 min; the supernatant was collected and stored at - 7 0 ° C until use. Purification of the substrates. The substrates of the enzymic reaction, H4MPT and coenzyme F,2o were obtained from M. thermoautotrophic'um by procedures modified from the or,¢s described in Refs. 12, 17 and 18. Since H4MPT is a quite oxygen-sensitive compound all steps during its f,urification were performed in an anaerobic glovebox. ,11 buffers and HaMPT-containing solutions were kept t.:der nitrogen atmosphere in serum bottles closed with '~lack butyl rubber stoppers. In a typical purification, ct.'lls (392 g, wet weight) were suspended in 30 mM sex;Jam acetate bu'ffer (pH 4) containing 1 mM dithiothrel ol to a final vol:,me of 800 ml. The ~uspension was I"laced in a boiling waterbath for 1.5 h and, after coolini~,, centrifuged at 18000 x g for 30 min. The supernatan: (560 ml) was decanted and applied to a column (20 x 4.8 cm) packed with DEAESephadex A-23 (acetale form) and equillb~',~ted ;n the above-mentioned sodium acetate buffer. The column was washed with 150 ml equilibration buffer and subsequently eluted with a linear gradient (1200 ml) of 160300 mM NaCI in the buffer followed by a wash with 1 M NaCI; 7-ml fractions were collected. HdMPT was detected enzymically as described below; the compound was present in the fractions eluting between 180-220 mM NaCI. Coenzyme F,~2o was eluted at I M NaCI. To the H,,MPT-containing pool (75 rid) NaCI was added to

a final concentration of 2 M, and H.~MPT was further purified by use of a Bonded Phase-Octadecyl (40 ,am) column (19 x 1.8 cm). The column had been activated with 100 ml methanol, washed with 100 ml 20 m M potassium phosphate (pH 5) and equil;!,ia,ed with 2 M NaCI in this buffer (100 ml). After applit, ation of the sample and washing with 100 ml equilibration buffer separation was performed with a li.aear gradient (600 ml) of 0-20% methanol in the phosphate buffer: 1 l-ml fractions were collected. H a M P T was eluted between 12-16% methanol and pooled (140 ml). For a final purification the pool was brought on a second DEAESephadex A-25 (CI- form) column (9 × 2.6 cm) equilibrated in 20 mM potassium phosphate buffer (pH 5). Separation ,~,as achieved with a linear gradient (60(J ml) of 0-500 mM NaCI in the phosphate buffer and 9-ml fractions were collected. H 4 M P T eluting between 370450 m M NaCI was pooled and concentrated by rotary evaporation at 30°C. The compound showed an ultraviolet spectrum in accordance w;'h the one previously repor!ed [3] and HPLC analysis showed only a single peak. Fractions containing coenzyme F4z0 were collected from the first DEAE-Sepha,a~,x A-25 column. The compound was detected oli the oasis of its characteristic blue fluorescence and its R F values upon thin-layer chromatography [17]. The poor was concentrated by rotary evaporation and desahed by use of Sep-Pak CIs cartridges [17 I. The desalted sample was applied to a second DEAE-Sephadex A-25 (CI- form) column (20 × 1.8 cm) equilibrated in 20 m M potassium phosphate bnffer (pH 8). After washing with equilibration buffer separation was achieved isocratically with 250 m M NaCI in the pho~ph~*e bt~ffer. Coenzym¢ F420 was pooled, concentrated by flash evaporation and des-~ted as described above. The compound was judged to be pure on the basis of its ultraviolet-visible light spectra [18] and HPLC analysis gave only one peak. Reduced coenzyme F420 (coenzyme F420H 2 ) w a s prepared in 89% yield from the oxidized form by reduction with sodium borohydride and purified as reported previously [12]; samples contained 7% of the oxidized species.

Purification of 5. IO-methylene-H, MPT dehydrogenase from M. thermoautotrophicum. Since the enzyme was found to be oxygen stable, all steps after the preparation of ceU-free extracts were carried out under aerobic conditions at 4°C. A "l solvents and enzyme preparations contained 0.5 m M CHAPS. Step 1. A m m o n i u m sulfate fractionation. To 10 ml cell-free extracts 15 ml 100% saturated a m m o n i u m sulfate was added overnight under gentle stirring. The solution was centrifuged for 15 min at 50000 × g and the supernatant, which contained the dehydrogenase, was carefully decanted. Step 2. Phe-~yl-Sepharose. The supernatant (22.5 ml) was brought on a column (36 × 1.7 cm) packed with

phenyI-Sepharose CL-4B and equilibrated in 2 M NaCI in 20 mM potassium phosphate bt, ffer (pH 6). After application of the sample the column was washed with 2 bed volumes of the equilibration buffer and separat~n was pursued with linear gradients of 2 0 M NaCI (400 ml) and of 0 to 25% (v/v) ethylene glycol (200 ml), respectively, both in lhe phosphate buffer. The enzyme was collected in a total volume of 94 ml in the fractions eluted between 6-20% ethylene glycol. Step 3. WP-PEI FPLC. The dehydrogenase was brought to homogeneity by FPLC using the Perkin-Elmer system equipped witla a Baker WP-PEI columa (11.5 × 1.3 cm). Equilibration and elution were performed with 20 mM potassium phosphate buffer (pH 6) at a constant flow rate of 0.6 m l / m i n under a pressure of 300 kPa: the eluate was monitored at 280 nm and 1.6-ml fractions were collected. After application of the enzyme the column was washed for 10 rain with the equilibration buffer followed by a 90-rain linear gradient of (:-2 M NaCI and a straight wash with 4 M NaCI. both in the phosphate buffer. The dehydrogenase was eluted during the latter step. Portions of the pool were concentrated and desahed by extensive washing with phosphate buffer on an Amicon PM 30 filler. Enzyme assays. By routine the dehydrogenase activity was measured spectrophotometrieally in the direction of 5,10-methylene-H4MPT oxidation and ccenzyme F~,0 reduction essentially according to [3,10]. The substrat.. 5,10-methylene-H4MPT is formed non-enzymieally from H4MPT and excess formaldehyde [4,51. The course of the reaction was followed by recording the increase in the absorbance at 335 nm and the decrease at 420 nm as a result of 5,10-methenyI-H4MPT (~335 = 22.4 mM • c m - ' , [10]) production and coenzyme F,20 (~4z0 -- 24.5 m M - ~• cm- t at pH 6) reduction. ^-'ivities were calculated on the basis of 5,10-methenyl-, 4MPT formation; the AA335 was corrected for the increase of the absorbance at this wavelength resulting from coenzyme F42oH2 (~a35 = 8.1 m M - a . e r a - i i101). 1 unit is defined as 1 /xmol 5,10-methenyI-H4MPT formed per rain. Reaction mixtures were prepared in cuvettes placed inside the anaerobic glove box (97.5% N,/2.5% H.,). The reaction mixtures (2 mi) contained 78 nmol H4MPT, 5 lamol formaldehyde and 87 nmol coenzyn~e F4z0 in 20 mM potassium phosphate buffer (pH 6) containing 1 M NaCI. After closing with rubber stoppers the cuvettes were taken outside the glovebox and placed in a thermostatted cuvette holder of the spectrophotometer. After a 5-rain preincubation at 600C the reaction was started by the anaerobic addition of 10-50 pl enzyme solution by means of a gas-tight syringe. During its purification the presence of H4MPT in the colum~ fractiens was tested after an enzymic conversion to its oxygen-stable derivative, 5.10-mcthenylH4MPT. Therefore, two serics of reaction mixtures in 10-ml serum bottles were prepared. The first (blank)

series contained 2 ml 20 mM potas:,ium phosphate bufler (pH 6). 78 nmol coenzyme Fa,o. I M NaCI and 5 p.mol formaldehyde, in the reaction mixtures of the second .,,cries extra dehydrogenase was present. 100-/~1 aliquots of the different column fractions were added to a vial of either series. After closing the vials with rubber stoppers and aluminum crimped seals, the serum bottles were incubated at 60°C under nitrogen atmosphere for an appropiale period of time. opened and the difference in the absorption at 335 nm between the respective vials of both series was determined. The column fractions showing a net absorbance at that wavelength contained H,MPT. .4nal.rtWal procedures. HPLC analyses were performed as described previously [12.17] using 40 mM sodium formate (pH 3.0) and a 0-25% linear gradient in methanol as solvent systems. Recording of ultravioletvisible light spectra and the spectropho~.ometric a~says took place either on a Hitachi U-3200 spectrophotometer or on a Hewlett-Packard 8452 diode array spectrometer connected to a Vectra ES/12 computer. The spectrophotometers were equipped with thermostatted cuvett,: holders. Enzyme concentrations were determined by the Coomassie brilliant blue G-250 protein binding assay 1191: boviae serum albumin setwed as the standard. Polyacrylamide gel electl, .'horesis (PAGE) was performed according to Laemmli [20] and proteins were stained with Coomassie brilliant blue. The molecular weight of the native protein was estimated by the method of Hedrick and Smith [21] on the basis o r the relative mobilities after electrophoresis on slab gels with 8, 9, 10 and 11 .% acrylamide. The following proteins were taken as standards: a-amylase (45000), alkaline phosphatase (100000) and the monomer (68000), dimer (136000) and trimer (204000) of bovine serum albumin. The molecular weight of the SDS-denaturatcd protein was determined by the method of Laemmli [201; cytochrome r (12 500;, a-chymotrypsin (25 100), carbonic anhydrase (30000), ovalbumin (43000), albumin (68000) and phosphorylase b (94000) served as the standards. Materials. Unless otherwise stated, chemicals were of the highest grade available. CHAPS, FAD and FMN were from Boehringer (Mannheim, F.R.G.). NADP ÷, NADPH, NAD ÷. N A D H and N,N,N',N'-t~tramethylethylenediamine (TEMED) were purchased from Sigma Fine Chemicals (St. Louis, MO). N, N'-methylene bisacrylamide (BIS), SDS. and ammonium persulfate were from Bio-Rad Laboratories (Richmond, CA). Serva Blue G for the protein determination was obt;~ined from Serva Feinbiochimica (heidelberg, F.R.G.), benzy! v;.ologen was from BDH Chemicals (Poole. U.K.). PhenyI-Sepharose CL-4B, DEAE-Sephadex A-25 and the molecular wei.,;ht calibration kits were bought .from Pharmacia Fine Che.'nicals (Uppsala, Sweden). WP-PE! FPLC column material, 40 txm

80 Bonded-Phase Octadecyl Cls and methanol ( H P L C grade) were from J.T. Baker (Deventer, The Netherlands). The coenzyme !=42o derivatives F*. Fo and F39o were a gift from S.W.M Kengen from our laboratory.. Gasses were obtained from Hoek-Loos (Schiedam, The Netherlands) and were made oxygen-free by passage over catalysts k i n d b given by BASF (Ludwigshafen, F.R.G.~ Prereduced R 3 - ; i wa~ us,:d at 150~C for nitrogen and RO-20 at amblcnt temperature was employed to rcmo,,e traces of ,,%'gen from hydrogen-containing gases.

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Results

Purificatwn and properties of 5, lO-methylene-H 4 M P T dehydrogenase from M. thermoautotrophicum The dehydrogenase was purified to a specific activity of 736 ttmol 5,10-methylene-H4MPT oxidized per min per mg in a three-step procedure as summarized in Table I. Since n o n d e n a t u r a t i n g polyacrylamide gel electrophoresis at this stage gave only one band (Fig. 1). the enzyme was j u d g e d to be pure. The procedure included a 60% a m m o n i u m sulfate fractionation, at which the enzyme is present in the supernatant, followed by hydrophobic-interaction c h r o m a t o g r a p h y and fast-protein liquid chromatography. Introductory. experiments had shown that the enzyme was oxygen-stable. Hence, all steps were carried out under aerobic condttions. Preliminary purification, however, was accompanied with severe and irreversible losses in activity. The losses were prevented when ionic or non-ionic detergents were included in the buffers and enzyme solutions. The zwitterionic detergent C t l A P S (0.5 m M ) proved to be most suitable. When kept in its presence, no loss in activity was found for the dehydrogenase upon storage at 4 ° C for more than 6 months. A remarkable aspect in the purification scheme (Table 1) is the 190% recovery, which is a result of the enzymic assay employed. As can be seen from the Table an a p p r o x i m a t e 2-fold enhancement in yie!d is observed after the phenyI-Sepharose step. During this step the d e h y d r o g e n a s e is s e p a r a t e d from 5,10-methylene-

~Front Fig. 1. Native polyacrylamide gel electrophoresis of purified 5,10methylene-H4MPT dehydrogenase. 10.6. 21.2 and 42.4 p.g enzyme were subjected to electrophoresis on 10% slab gels and subsequently stained with Coomassie brilliant blue as described under Materials and Methods.

H4MIY'[', reductase [12]. W h e n both enzymes are present in the assay, they c o m p e t e for 5 , 1 0 - m e t h y l e n e - H 4 M P T as a c o m m o n substrate; the reductase uses c o e n z y m e F420H 2 produced in the d e h y d r o g e n a s e reaction as the r e d u c t a m (reactions 1 a n d 2). The result is a disproportionation of 5 , 1 0 - m e t b y l e n e - H 4 M P T into a b o u t

TABLE i

Purificationof 5,iO-methylene-II,MPT deh),drogenasefrom M. thermoautolrophicum The purification was started from l0 ml crude cell-free extract of M. thermoautotrophicum. Activities were measured in the direction of 5,10-mcthylene-H4MPT oxidation as described under Materials and Methods. Units are t.xpresscd as lamol 5,10-methenyI-H4MPT formed per rain. Step

Crude extract

Total protein (rag) 390

Total activity (units) 1900

Specific activity (units/rag) 4.87

Factor (-fold) 1

Recovery (%) 100

Ammonium sulfate (60% supcrnatant)

99

1800

Phcnyl-Sepharose

11.I

3400

304

62

185

4.8

3500

736

151

191

FPLC WP-PEI

18.24

3.7

95

/

~"15

/

/, 1:[

\

/

\ \\

/

\

/

o

~o

\

/

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slope .qm Fig. 2. Determination of the molecular weights of natLvq and SDS-dcnaturated 5.10-m~:th~.lt.nc-H4MP'rtiehydrogenase. IA) Dehydrogenase (o) and enzymic standards (o) of known molecular weighl~,a~, specified under ,Ma~etaalsand .Mczht~ were ¢lectrophorised on 8. 9. 10 anti llCf slab gels and the relative inabilities (R m) were mea.~ured. Apparent molecular weight.,: are plcttc.t/ as a function of the slope-,, derived from the 100 log ]00x R m vs. ~ polyacrylamidc curves. (R) SDS-zrealed dehydro~;cna~c~c,~ was subleczcd to SDS-eleczlophorcsis in a 13% gel containing 0.1% SDS together '~lth the molecular standards (O) as specified under Materials and Mcthc
e q u a l a m o u n t s o f 5 , 1 0 - m e t h e n y I - H 4 M P T a n d of 5m e t h y l - H 4 M P T , w h e r e a s n o net r e d u c t i o n of c o e n z y m e F42o occurs. T h i s w a s s u b s t a n t i a t e d b y H P L C a n a l y s e s o f the r e a c t i o n m i x t u r e s a n d in s p e c t r o p h o t o m e t r i c det e r m i n a t i o n s r e c o r d e d s i m u l t a n e o u s l y at 335 a n d 420 n m . In a d d i t i o n , w h e n e n z y m e p r e p a r a t i o n s w e r e used free o f the r e d u c t a s e , the final a b s o r b a n c e at 335 n m w a s 2-fold h i g h e r a n d c o e n z y m e F420 w a s r e d u c e d stoic h i o m e t r i c a l l y with respect to 5 , 1 0 - m c t h e n y I - H 4 M P T f o r m a t i o n (see below). D u r i n g the p r e p a r a t i o n o f the cell-free extracts all d e h y d r o g e n a s e activity w a s p r e s e n t in the s u p e r n a t a n t o f t h e brokezt cells a f t e r c e n t r i f u g a t i o n . T h e e n z y m e w a s p u r i f i e d b y a f a c t o r 151 ( T a b l e 1). Since the specific activity o f the cell-free e x t r a c t s m a y be u n d e r e s t i m a t e d b y a f a c t o r 2 for r e a s o n s given a b o v e , the a c t u a l purific a t i o n f a c t o r is a b o u t 75. T h i s implies t h a t the d e h y d r o g e n a s e c o m p r i s e s a p p r o x . 1.5% o f the total soluble p r o t e i n s , w h i c h a g r e e s w i t h its c e n t r a l m e t a b o l i c role. A n a p p a r e n t M r o f 2 1 6 0 0 0 c o u l d b e e s t i m a t e d for the n a t i v e d e h y d r o g e n a s e (Fig. 2A). D c n a t u r a t i n g SDSp o l y a c r y l a u l 6 d e g a v e o n e b a n d with a n M, o f 3 6 0 0 0 (Fig. 2B). T h i s indic~t.es t h a t the e n z y m e is a h e x a m e r o f identical s u b u n i t s .

Substrate specificity and reversibility of the reaction By r o u t i n e the d e h y d r o g e n a s e w a s d e t e r m i n e d in the direction of 5,10-methenyI-H4MPT formation and

c o c n z y m e F~20 r e d u c t i o n . A typical e a a m p l e o f the s p e c t r o p h o t o m e w i c a s s a y is s h o w n in Fig. 3. N o react.on w a s obser~.ed in the a b s e n c e of c o c n z y m e F42o. In the c o u r s e of the r e a c t i o n the a b s o r b a n c e at 335 n m increased as a result o f 5 , 1 0 - m e t h e n y I - H 4 M P T a n d

Absorbance

1.2

~_~2m_. i/

335n,m __.

/ . . . . .

.s

time[~in )

Fig. -,. 5,)0-Meth)Iene-H4MPT oxidation and cocnz]m~ 1=42o reduction by purified 5,10-methylcne-H4MPT dchydrogenase_ The spectraphotometric assay contained 19.5/~M 5.I0-methylene-H,sMPT and 25 ~M coenzyme F4.,o and was performed as de~'nbed under Materials and Methods. The f2action was started after about 2 rain by the anox/c addition of 0.! it8 purified enzyme wi;.h a specific activity of 736 units/mS.

82 c o e n z y m e F,~,uH," f o r m a t i o n . Simultaneously. the abs o r b a n c e at 420 n m decreased d u e to the r e d u c t i o n of c o e n z y m e F4~o. F r o m the m o l a r ab+';,orbances given under Materials a n d M e t h c d s a n d ' h e differences between :.rte initial a n d final a b s o r p t i o n s o n e m a y calculate t h a t 18.5 p.M 5 , 1 0 - m e t h e n y I - H 4 M P T w a s formed, w h e r e a s the c o n c e n t r a t i o n of c o e n z y m e F420 h a d d e c r e a s e d b y 17.5 p.M. T h e d a t a arc in a g r e e m e n t with the 1 : 1 s t o i c h i o m e t r y of reaction (1). 3ince 19.5 p M meth'AeneH + M P T was present at the start, u n d e r the e x p e r i m e n tal c o n d i t i o n s this s u b s t r a t e is Jlearly q u a n t i t a t i v e l y c o n v e r t e d into 5 , 1 0 - m e t h e n y l - H 4 M P T . W h e n at the e n d c o e n z y m e F420H2 w a s a d d e d a n a e r o b i c a l l y , the abs o r b a n c e at 420 a n d 335 a m increased a n d ,Jccreased, respectively ( d a t a n o t shown), i n d i c a t i n g t h a t the o p p o site reaction also t say take place, T h u s , 5,10-methyleneH4MPT oxidalion and 5,10-methenyI-H4MPT reduction are essentially reversible [5,10]. C o e n z y m e F42n is a quite specific c o - s u b s t r a t e . N a t u ral electron carriers, like N A D ( P ) + a n d the flavins F M N a n d F A D , as well as the artificial el,:ctron c a r r i e r benzyl viologen, were inactive. N A D H a n d N A D P H also did not fut:ction as r e d u c t a n t s in the e n z y m i c r e d u c t i o n of 5 , 1 0 - m e t h e n y I - H a M e l . C o e n z y m e Fa20' however, c o u l d be r e p l a c e d by its derivatives F + a n d Fct, w h i c h lack the l a c t y l g l u t a m y l a n d the 5 ' - p h o s p h o r y l l a c t y l g l u t a m y l residues of the i n t a c t eoenzyme. C o m p a r e d to c o e n z y m e F42o, tested in 42 ~,M c o n c e n t r a t i o n , activities in the presence o f F + (40 p.M) a n d F 0 (40 # M ) were 66 a n d 117%, respectively. In c o n t r a s t . F39o, 8 - O H a d e n y l y l a t e d F420, w a s inactive. F39o is p r o d u c e d f r o m A T P a n d c o e n z y m e F4:o [221, w h e n g r o w i n g cells [231 o r cell extracts [22] of methano~'c;,i~, bacteria, like M. t h e r m o a u t o t r o p h i c u m , are e x p o s e d to o x y g e n . A b o v e , we pointed to t'.'. reversibility o f r e a c t i o n (1). In the following e x p e r i m e n t it w a s tried to assess the e q u i l i b r i u m c o n s t a n t . Therefore. a n u m b e r o f a s s a y s were p e r f o r m e d c o n t a i n i n g v a r i o u s initial c o n c e n t r a tions of 5 , 1 0 - m e t h y l e n e - H 4 M P T , c o e n z y m e F420 as well as its r e d u c e d form, c o e n z y m e F420H2, a n d p u r i f i e d d e h y d r o g e n a s e ( T a b l e Ill. W h e n the r e a c t i o n s h a d rea c h e d the e q u i l i b r i u m , the c o n c e n t r a t i o n s of the rea c t a n t s were d e t e r m i n e d . F r o m the d a t a listed in T a b l e II o n e m a y c a l c u l a t e a Keq = 2.24 × 107 M -! at 6 0 ° C for the reaction (3). 5,10-methylene-H4MPT + coenzyme F4:0+ H + 5.10-methenyI-H4 MPT + coenzyme

F+~oH ~

(3)

Kinetic properties a n d effectors o f the e n z y m i c reaction T h e d e h y d r o g e n a s e reaction s h o w e d a d e f i n e d temp e r a t u r e o p t i m u m at 6 0 ° C , situated n e a r the o p t i m a l g r o w t h t e m p e r a t u r e ( 6 5 ° C ) o f M. t h e r m o a u t o t r o p h i c u m . A p H o p t i m u m w a s o b s e r v e d at p H 6.0 for the r e a c t i o n followed in the direction of m e t h y l e n e - H 4 M P T o x i d a tion.

TABLE 11 Determination of the equilibrsum constant of the 5. I0 raeth.vlene-H, An,PT dehrdrog(,no.sc reaction Reactions were measured in Ihc direction of 5.10-methylene-H4MPT oxidation at pH 6.0 ([tI * ]~ 10 -6 M) as described under Materials and Methods in the presence of 01 ~g p:,rif!ed '-'n~yme and inUl:d corlcentrallt ns of sub~,trates a~ given m the Table. Final concentrations of coenzyme F4:0 and of 5.10-mether,yi-H ~MVr were calculated from the ahsorbancies at 40i and 335 nm. respectively', at the end of the rchctson. In the latter case the contribution of coenzymt. F,I2oH2 at this wavel.':ngth was c~rrected for. (CH, = ~ 5.iO-rr+ethyh.ne-H,+MPT: ++CH' = I. 5,10-metbenyI-H,+Ml'T. Expcr- Concentration irr~ent

K~ x l0 -<' (M - I ) J

(pM)

No.

F+2o

F42(,H: (t'H~ = ) (CH + = )

1

initial 40.5 3 9 firtat 4.9 38.6

48.8 13.2

0 35.6

21.6

2

initial 54.3 17.1 final 13.3 57.9

48.8 8.0

0 40.8

21.9

3

initial 68.5 37.7 final 25.2 81.0

48.8 5.5

O 43.3

25.3

+ K~: = ([CH" = ).[Es.+.oH.,I)/(ICH+= HF,2ol'IH " I)

High concentrations of monovalent cations markedly s t i m u l a t e d th,. r e a c t i o n in the a p p a r e n t o r d e r K + < C s * < N a < NH.~ < L i " ( T a b l e iII). T h e s t i m u l a t i o n w a s e n h a n c e d w h e n the salt c o n c e n t r a t i o n s were f u r t h e r increased: 3 M N a C I , for instance, s t i m u l a t e d a b o u t 5-fold. M g 2* s t i m u l a t e s the 5 , 1 0 - m e t h e n y I - H 4 M P T c y c l o h y d r o l a s e f r o m M . f h e r m o a u t o t r o p h i c u m [8] a n d the ion is r e q u i r e d for the N A D * - d e p e n d e n t 5 , 1 0 - m e t h :/lene-H4folate d e h y d r o g e n a s e f r o m Ehrlich ascites t u m o r cells [24]. M g 2 . (10 r a M ) o r o t h e r b i v a l e n t cations, like M n 2. (5 m M ) o r Z n 2+ (5 raM), h o w e v e r , h a d n o effect o n the d e h y d r o g e r , ase re,~ction, w h e r e a s E D T A (10 r a M ) w a s not i n h i b i t o r y . T h i s L:.plies t h a t bivalent c a t i o n s are n o t r e q u i r e d for e n z y m a t i c activity.

TABLE III Effect of monovalem ¢atwns on the 5.lO-met/Tylene+H+MPT deh.vdr~ genase/ronl M. lhermoautotrophicum

Reactions were followed in the direction of 5.10-methylene-H4MPT oxidation as described under Materials and Methods in the presence of 25 #M coenzyme F4,0, 19.5 :~M 5.10.methylene-H4MPT, 0.l p$ protein in 20 mM potassium phosphate buffer (pH ~..0) and I M salts as indicaled. The relative (v.t¢ of the reaction withoat additional salt was sel at 100~ and ~:qualed a rate of 22.4 nmol/min. Salt

Relative rate (%)

-

100

L.iCl NH4C1 NaCI KCI CsCI

415 340 300 175 250

The 5,10-methylene-H.~MPT dehydrogenase reaction is in two respects comparable with 5.10-methyleneH4folate dehydrogenase: H4MPT is a structural and functional analogue of H4folate [51 and cocnzvme Fa.. act.,, in hydride transfer and is as such functionally related to the nicotinamide cofactors [25]. NADP-dependent 5.10-mcthylene-H4folate dehydrogcnase from Clostridium cylindrosporurn and Salmonella tvphimurium are regulated by ATP [26]. This compound t'sted at 1.25 mM, both in the prese,~c ~nd abseace of Mg -~: (2.5 raM) did not affect the dehydrcgeaz=e from M. thermoautotrophicam. Neither did glycine or cysteine at concentrations of 0 to 2.5 mM influence the reaction. As mentioned above. NAD or NADP were not substrates of the methanogenic dehydrogenase, 1 mM concentrations of these compounds, in addition, did not act as inhibitors. These and the above-mentioned potential effectors were tested in reactions in the directicn of 5,10-methylene-H4 MPT oxidation.

1 #v( rn,n nn~)l'l I

1 tapfl K'~ilxim,n r 'not'~ ;

l h e st.eady-,~tatc enzyme kinetics were investigated by measuring the initial velocities at various 5.10-methylene-H~MPT and coenzyme F~2,, concentrations. Reciprocal (Lineweaver-Burk) plots are presented in Fig. 4A and C and show a series of non-parallel straigh: lines, which intersect the respective abscissa at a single point left of the ordinate. The patterns ,are indicative of a teraar3, complex (ordered) mechanism This ~.onc!usion was ,~ubstantia~ed by non-linear regression analysis cf the experimental data by the use of a PC enzyme kinetics pr,)gram [27]. From the iutercept with the abscissa one may calculate an apparent K m = 33 #M for 5.10-methylene-H.tMFT (Fig. 4C) and a K~, = 65 ~tM for coenzyme Fa,0 (Fig. 4A). RcFlots of the reciprocal apparent V,,~, values obtained from Fig. 4A and C vs. the reciprocal concentrations of 5,10-methylene-H4MPT and of co,enzyme F~:,o, respectively, again yielded straight lines (Fig. 4B and D). From the respective intercepts with the abscissa, apparent Km values of 33 and 65 #M for 5.10-methylene-HaMPT and coenzyme F4.,ocould be calculated in agreement with the above-given data. Both from the Fig. 4B and C an identical apparent V,,~ = 480 n m o l - n u n ~ is determined, which eqti,als a maxtmal specific activity of 4000 btmol - rain I. rag- t protein. DL~cu,~,sion

io

ioio' ~

to k

~

~o ,'~

llacpVma, lm,n nmo~ ?

7='"' ,,/,,L ,4° 4 .9" ii,/ ' ~ ~ ~o ~ ~i

1/mtll~ltl~ itl fill i # M -11

/-7o'

~o' ~o' i

i#lUOl~14"ll

Fi1. 4. Lincwe~ver-Burkkinetic plot~ of the .$,10-methylcne-H,iMll'f dehydrogena~ ¢¢action. The spectropholomcmc a.,.say~, were performed in the direction of 5.lO-rnethylene-H.,MPT oxidation as described under Matenal~ and Methods i, the pc~cnce of 0.12 ti& ourified e~tzyme.(A) The reciprocal initial vclocitic~ of the re.actions are plotted vs. reciprocal c.~¢l~zym~F4~o con~,,ntrations in the .~r~-~cnt,'e of 5,10-methylene-H4MPi" as indica:ed. ~C) Reciprocal ,"¢acuon rates ate plotted as a function of reciprocal 5,iO-methylene-H4MFq" cort~entration~lU the indicated concentrationsco'~)zyme F~20 (B) and (D) represent the double-reciprocaJ plo;s of the apparent V~ ~alues obtained from Fig. 4A and C vs. the concentrationsof 5,10-r~ihylen¢-H4MPT and of coin:yl~ F.ilo,respicuv¢ly.

In this paper we have presented ~t ra',her simple procedure, which yields with nearly complete recove~ ilomogent,otls 5,10-methylene-Fi4MPT dehydrogenase from M. thermoaurotroohicum strain AH. Previou31y, lbe enzyme had been purified from the J H [10] and Marburg [I1,13] strains of the o, ganism, though with lower yield a n d / o r lower specific activity It was foand here that the presence of a suitable detergent was a prerequisite foi maintenance of the actwity of th~ otherwise oxygen-st=bit protein. The bchaviour during purification and the properties of the present enzyme clearly differ from those reported for formylmethanofuran : H tMPT formyltransferase [7|, 5,10-methenyI-H4MFf eyclohydrolase [8.11] alid coenzyme ~zo-dependent 5.10-methylene-H4MPT reductase [12] from the same o~anism. Moreover, apatl from the 5,10-methylene-H4MFq" oxidation and 5A0-methenylH4MPT reduction no other enzymic activity could be attributed tc the dehydroget~ase. This sugg~ts that the ¢lizyme. like the other H.IMPT-dcpendcnt proteins mentioned, is a monofunction',d enzyme. The dehydrogena~ from M. thermoautotraphicum strain Marburg that was purified under anoxic conditions by about the same factor and to the same specific activity though in !,~wer yield (10,~) behaved as an hydrogenase, since protons and hydrogen, rather than coenzyme F,20, were reported io be involved as substrates ~13|. Our aerobically purified enzyme did not show such activity aad

84 the latter c o e n z y m e or its derivatives F + a n d Fo were strictly required for enzymic activity. T h e purified d e h y d r o g e n a s e h a d a specific activity of 736 p.mol 5 , 1 0 - m e t h y l e n e - H 4 M P T oxidized per rain per m g protein. F r o m p o l y a c r y l a m i d e gel electrophoresis it was c o n c l u d e d that the e n z y m e is a p p a r e n t l y a h e x a m e r of six id~zatical 36 k D a subunit'~. F r o m the a p p a r e n t m o l e c u l a r weights a n d Vm~~ values o n e m a y d e t e r m i n e a lurnovel n u m b e r o f 2400 s e c - ~. Ultraviolet-visible light s p e c t r a of the purified e n z y m e ( d a t a not s h o w n ) did n o t point to the presence of (an) a b s o r b i n g p r o s t h e t i c group(s). S t e a d y - s t a t e kinetics indicated t h a t the enz y m i c reaction o c c u r s b y a t e r n a r y c o m p l e x m e c h a n i s m . C o e n z y m e F420 acts in h y d r i d e transfer [25]. T h u s . a t e r n a r y c o m p l e x w e c h a n i s m , then. is in a g r e e m e n t with a direct transfer of such g r o u p b e t w e e n b o t h substrates. At the e x p e r i m e n t a l c o n d i t i o n s ( p H 6.0) the o x i d a tion of 5 . 1 0 - m e t h y l e n e - H 4 M P T is s t r o n g l y f a v o u r e d (see Ref. 10 a n d this paper). However, at n e u t r a l p H 7 the reaction in the direction of 5 , 1 0 - m e t h e n y I - H , M P T r e d u c t i o n (reaction 1) b e c o m e s m o r e feasible h a v i n g a Keq = 0 . 4 4 - 107 M. F r o m this value a n d f r o m the midp o i n t potential E~ = - 3 5 0 mV [25] for c o e n z y m e F420 o n e m a y calculate a n Et~ = - 3 6 2 mV for the r e d o x couple 5,10-methylene-H 4M PT/5,10-methenylH 4 M P T . U n d e r h y d r o g e n a t m o s p h e r e at 6 0 ° C the overall reaction (4) in the physiological direction o f 5,10m e t h e n y I - H 4 M P T r e d u c t i o n will be s o m e w h a t exergonic ( A G ' = - 10.1 k J / m o l ) . H z + 5.10-methenyI-H4MPT ~ 5.10-methylene-H4MPT+ H ÷

(4)

Active t r a n s p o r t of s o d i u m ions is k n o w n to p l a y a n i m p o r t a n t role in m e t h a n o g e n i c e n e r g y m e t a b o l i s m at the r e d u c t i o n levels between C O z a n d 5,10-methyleneH 4 M P T [28-301. S o d i u m ions p r e s e n t in high c o n c e n t r a t i o n s m a r k e d l y s t i m u l a t e d the d e h y d r o g e n a s e reaction. T h e c a t i o n was, however, not specific in its a c t i o n and, in fact, the reaction also p r o c e e d e d u s i n g e n z y m e a n d s u b s t r a t e p r e p a r a t i o n s purified in the a b sence of s o d i u m ions. A f t e r b r e a k a g e of the cells a n d s u b s e q u e n t c e n t r i f u g a t i o n all activity was recovered in the s u p e r n a t a n t , w h i c h indicates t h a t the d e h y d r o g e n a s e is a soluble protein. These a r g u m e n t s d o not s u p p o r t a d i r e c t function for the e n z y m e in the m e t h a n o g e n i c e n e r g y m e t a b o l i s m between the C O 2 a n d m e t h y l e n e ~'eduction level. Acknowledgement T h e w o r k of J.T. Keltjens w a s m a d e possible b y a senior fellowship f r o m t,he R o y a l N e t h e r l a n d s Society of A r t s a n d Sciences ( K N A W ) .

References 1 Rou,.'i,~re.P.E. and Wolfe. R.S. O988) J. Biol. Chem. 263. 7913 7916. 2 Keltjens. J.T. and Van der Drift. C. (1988) FEMS Microbiol. Rev. 39. 259-303 3 Escalante-Semerena. J.C.. Leigh, J.A.. Rinehart. K.L.. Jr. and Wolfe. R.S. (19811 Proc. Natl. Acad. Sci. USA 81, 1976-1980. 4 Escalante-Semerena. J.C.. Rinehart. K.L.. Jr. and Wolfe. R.S. (19811 J. Biol. Chem. 259. 9447-9455. 5 Keltjens. J.T. and Vogels. G.D. (19881 BioFactors 1.95-103. 6 Van Beelen. P.. Van Neck, J.W.. De Cock. R.M.. Vogels. G.D.. Guyt. W. and Haasnoot. C.A.G. (19811 Biochemistry 23, 44484454. 7 Donn¢lly. M.I. and Wolfe. R.S. (19861 L Biol. Chem. 261. 1665316659. 8 DiMarco. A.A.. Donnelly. M.I. and Wolfe. R.~. (19851J. Bacteriol. 169. 1372-1377. 9 Te Br~mmelstroet. B.W.. Hcnsgens. C.MH., Kelqens. LT.. Van der Drift. C. and Vogels, G.D. (19901 J. Bacteriol. 172..564-571. 10 Hartzell. P.L.. Zvilius. G.. Escalante-Semerena. J.C. and Donnelly. M.I. (1985) Biochem. Biophys. Res. Commun. 133. 884-890. II Mukhopadhyay. B. and Daniels, L. (19891 Can. J. Microbiol. 35. 499- 507. 12 Te BrOmmelstroet. B.W.. Hensgens, C.M.H.. Kelqens. J.T., van der Drift. C and Vogels. (3.D. (19901 J. Biol. Chem. 265. 18521857. 13 Zirngibl. C.. Hedderich. R. and Thauer. R.K. (1990) FEBS Lett. 261. 112-116. 14 Brandis. A.. Thauer. R.K. and Stettcr. K.O. (19811 Zbl. Bakt. Hyg., I. Abt. Orig. C2. 311-317. 15 SchBnheit, P.. Moll. J. and Thauer. R.K. (1980) Arch. Microbiol. 127. 59-65. 16 Van Beelcn. P., Geerts. W.G.. Pol. A. and Vogels. G.D. (19831 Anal. Biochem 131. 285-290. 17 Keltjcns. J.T.. Cacrteling, G.C. and Vogels. G.D. (19861 Methods Enzymol. 122. 412-425. 18 Eirich. L.D., Vogels. G.D. and Wolfe. R.S. (19781 Biochemistry 17, 4583-4393. 19 Sodmak, J.J. and Gros.~berg. S.E. (19771 Anal. Bioehem. 79, 544552 20 Laemmli, U.K. 0970) Nature (Lond.) 227. 680-685. 21 Hedrick. J.L. and Smith. A.J. 0968) Arch. Bioehem. Biophys. 126. 155-164. 22 Kengen. S.W.M.. Kelljcns. J.T. and Vogels. G.D. (19891 FEMS Microbiol. Leu. 60. 5-10. 23 Kiener. A.. Orme-Johnson. W.H. and Walsh. C.T. ll9881 Arch. Microbiol. 150. 249-253. 24 Scrimgeour. K.G. and Huennekens. F.M. (19601 Biochem. Bidphys. Res. Commun. 2. 230-233. 2.5 Jacobson. F. and Walsh. C. (19841 Biochemistry 23. 979-988. 26 Uyeda. K. and Rabinowitz. J.C. (19671 J. Biol. Chem. 242. 43784385. 27 Perrclla, F.W. (19881 Anal. Biochem. 174. 437-447. 28 Sch~*mheit, P. and Beimborn. D.B. (19851 Arch. Microbiol. 142. 354-361. 29 Mtlllcr. V.. B:auL M. and Gonschalk, G. (19871 Eur. J. Biochcm. 162, 461-466. 30 Kacslcr. B. and SchOnheit. P. (19891 Eur. J. Biochem. 184. 223-232.