On the structure and linkage of the covalent cofactor of methylamine dehydrogenase from the methylotrophic bacterium W3A1

Vol. 141, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 562-568

December 15, 1986

ON I ~ { E S T R U C T ~ A N D L I N K A G E O F T [ E COVALF']'aT C O F A C T O R O F ~ L ' T H Y ~ N E D ~ E F R O H T ~ [ ~ - ~ I C B A C T ~ I U M W3AI William S. McIntire a'

and John T. Stults b

aveterans Administration Medical Center, Molecular Biolog~ Division (151-S), San Francisco, CA 94121 bMichigan State University, Department of Biochemistrv , East Lansing, MI 48824

Received October 30, 1986

Short amino acid sequences around the two linkage sites of the cofactor of methylamine dehydrogenase are presented. Mass spectral data indicates that the covalently bound cofactor is the tricylic pyrroloquinoline quinone (PQQ). However, the 3 carboxyl groups characteristic of this o-quinone are absent. A cysteine thioether, via a methylene bridge, and a serine ether link the cofactor to the small subunit of methylamine dehydrogenase. © 1986AcademicPress,Inc.

2,7,9-Tricarboxy-iH-pyrrolo[2,3-f]-quinoline-4,5-dione or quinone (PQQ)

is the novel redox cofactor for bacterial,

fungal enzymes (1,2,3). bacteria

( 2 ) , and

PQQ is also secreted by

stimulates

the

a

a

variety

quinoproteins. plasma

amine

of species.

that for plasma of PQQ,

an

oxidase,

and

methylotrophic as well as

essential

nutrient

Interestingly PQQ is covalently linked to several

Acid hydrolysis of bacterial methylamine dehydrogenase

(MADH),

kidney diamine oxidase (2), and mitochondrial choline

dehydrogenase. PQQ

of

plant,

growth of other bacteria (4),

dehydrogenase (3) released the covalent apoglucose

animal,

number

animal and plant cells (5). This suggests that PQQ is for

pyrroloquinoline

( 2 ) . Pepsin

The

cofactor

fluorescence

digestion

of

in

a

form

that

activated

spectra were also reminiscent of

2,4-dinitrophenylhydrazine

treated

amine oxidase released a small amount of the 2,4-dinitrophenylhydrazone as determined by the comparison of the

chromatographic

and

spectral

properties with those of the authentic derivative (6).

*To whom correspondence should be addressed. Abbreviations: PQQ, pyrroloquinoline quinone; MADH, methylamine dehydrogenase; FAB, fast atom bombardment; HPLC, high pressure liquid chromatography; ESR, electron spin resonance spectroscopy; ENDOR, electron nuclear double resonance spectroscopy.

0006-291X/86 $1.50 Copyright © 1986 by Academic Press, Inc. All rights qf reproduction in any Jorm reserved.

562

Vol. 141, No. 2, 1986

Work

on

MADH

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

bacterium

W3AI

fluorescence spectra of the denatured known

for

MADH from amino

PQQ

(7).

Ishii e t a ] .

Meth~lobacterium

acid residues.

sp.

indicated

cofactor

that

subunit

the

UV-visible

differed

from

and those

(8) presented evidence that the cofactor of

AM1 is

linked

via

two

distant

unidentified

One explanation for these observations would be that PQQ

is modified more dramatically than suggested by the accounts release from various proteins (2,3).

To test this hypothesis,

of

its

pristine

a mass spectral

analysis of the structure of the cofactor bound to MADH was undertaken. ~I~E~ITAL

F

~

Materials. All reagent were of the highest quality available commercially. MADH and methanol dehydrogenase from bacterium W3AI (N.C.I.B. 11348) were purified by the same procedure ( 7 ) . PQQ was isolated from the methanol dehydrogenase (9) and the growth media of this organism (2), and was purchased from Fluka Chemical Corp. as methoxatin. Methods. After 2 h, a solution of 95 mg (748 nmol) of MADH + 60 umol of semicarbazide in 5 mL of 1.44 M Tris, pH 8.75, was concentrated to 200 uL, and 4 mL of 6 M guanidine HCI were added. Under Argon, 20 uL of 2-mercaptoethanol were added, the mixture incubated for 3 h, then 300 mg of Na iodoacetate were added, and allowed to react for 3 h. The sample was diluted to 8 mL, and run on a 2.5 x 57 cm Bio-Gel A-0.5m (Bio-Rad) column in 2.5 M guanidine HCI + 0.2 M potassium phosphate, pH 7.0. The fractions containing the cofactor subunit were made 10% in acetic acid and applied to a 0.9 x 6 cm Florisil (Fisher Scientific) column, which was washed with 100 mL 5% acetic acid, then H20 to neutral pH, and the protein was eluted with 20% pyridine. The sample was lyophilized and dissolved in 2 mL of 50 mM morpholinopropane sulfonic acid + I0 mM CaC17, pH 7.0. The solution was incubated with 0.75 mg pronase at 37 0C. The digest-was monitored by chromatography on a 7.5 x 0.75 cm TSK DF_~-5PW column (Phenomenex). The gradient was 20 mM to 300 mM amunonium phosphate, pH 7.0, in 30 min (flow rate 1 mL/min and detection at 436 nm). The reaction was terminated after 6 h. This HPLC method was used to purify the major cofactor peptides from this digest. After acidification to pH 2.0, each peptide was further purified on the PepRPC HR5/5 reverse phase column (Pharmacia). The method involved a gradient from 100% water to 60:40 water/acetonitrile in 20 min (all solutions 0.1% in CF3COOH , flow rate 0.7 mL/min, detection at 436 nm). T h e samples were lyophilized, redissolved in a small volume of water (0.25 - 2.0 mL), and analytical HPLC by the methods just described indicate that the peptides were pure when detected at 214 nm. The major peptide was treated for 6 h with 1 mg aminopeptidase-M in 2 mL of 20 mM potassium phosphate, pH 7.2 and 37 °C, and was processed as described above. Following digestion with 1.5 mg carboxypeptidase-Y in 2.0 mL of 0.I M pyridinium acetate, pH 5.5 and 37 °C, for 3 h, the solution was lyophilized and the cofactor peptide purified by the same HPLC methods. Amino acid analysis was carried out using a published procedure (i0). The sequence analysis was done in the laboratory of Dr. A. J. Smith at the University of California, Davis. FQQ (500 nmol) was mixed with 5 umol semicarbazlde in 250 uL of 1% CF.COOH in H~O. After 1 h, the mixture was centrifuged, the semicarbazone was~ed 4 times-with 250 uL of 0.1% aqueous CF3COOH , and dried i n v a c u o . The mass spectra were obtained by the fast atom bombardment ( F A B ) with a JEOL JMS-HXIIOHF mass spectrometer, operated at 10 kV accelerating voltage and resolution = 1,000 or 3,000. The dried samples were dissolved in 5 uL of dimethylsulfoxide or water. Then 2-3 ~L of each solution were dissolved in approximately 1 uL of matrix compound on the FAB probe. The matrix was either

563

Vol. 141,No. 2,1986

BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS

glycerol, triethanolamine, or a 5:1 mixture of dithiothreitol/dithioerythritol. The latter two matrices gave the best spectra. The samples were bombarded with 6 keV Xe atoms. The conversion dynode of the postacceleration detector was operated at 20 kV. Data were acquired with a JEOL DA-5000 data system. Both negative ion FAB and positive ion FAB scan were obtained. Tandem mass spectrometry was used to analyze the "parent" ions, by performing a linked scan at a constant magnetic field/electric field ratio. Dissociative collisions of the parent ions with He atoms generate "daughter" ions which gave further valuable structural information. RESULTS

Fkass Spectral J~alysis of F ~ . enzymes

to

which

it

is

In the past,

non-covalently

analysis of PQQ extracted from

bound

or

from the growth media of

bacteria,

involved the reactivation of apoglucose dehydrogenase,

and/or a

comparison

PQQ (1,2).

However,

of

the

variant

fluorescence

spectra

with

that

HPLC analysis, of

authentic

forms might exists which have similar properties

to those reported for 2,7,9-tricarboxy-PQQ

(structure I).

H

Hence, mass spectral

COOH

.0%? 'F \\, HOOC''~N/ ~ ' 0 0

I data for various identification

PQQ

sample

could

be

were

collected

made.

These

so

that

studies

a

also

direct

structural

provided

reference

information needed for the analysis of the structure of the MADH cofactor. Commercial P ~

gave a mass for the parent ion (M-H)- = 331 by

and (H+H) + = 333 by positive FAB,

negative

yielding a molecular weight of 332.

2 mass units higher than expected for the o-guinone I.

FAB

This is

Gainor and Weinreb (ii)

reported a molecular weight 2 units higher for synthetic FQQ trimethyl ester as measured by electron impact mass

spectrometry.

reduced

by

to

the

dihydroquinone

from the growth media of bacterium W3AI and identical

with

that

guinone

appears

to

be

accepting hydrogen atoms from the matrix,

which results in an increase by 2 mass units.

were

This

The spectra,for the PQQ isolated from

the

methanol

obtained for the commercial sample.

semicarbazones for the commercial PQQ and that isolated from the

564

dehydrogenase Further, growth

the media

Vo1.141, No. 2,1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

also

which

is more p r o m i n e n t

form,

identical:

whereas

the

(M-H)- d a u g h t e r groups

(M+H) + = 388 earlier

as HCOOH and C02,

Amino

Acid

a~

peptides

derived

peptide,

which

accounted

3 - Set,

cofactor

binding

sites

experiments

detected

from MADH were o b t a i n e d for

from this analysis

aminopeptidase-M

4 - Set,

that

the

can

be

digest

established

ambiguity

A.

lose

oxidized (M+H) + a n d

of

the

COOH

Phe;

5 - Set(?);

AM1

the

with

(9). I.

released

2 - Pro;

6 - Val;

7 - Ala. small

a r o u n d the

result

to the cofactor

from

are not

of the peptides. at

degree

the of

2 Set and a

(part B of Chart

to the

An important

side of the Pro rather

major

to sequence

The sequences

analysis

high

The

Val;

of the cofactor

sequence

a

for s e l e c t e d

subjected

1 - Set,

amino acids a t t a c h e d

of this p e p t i d e

is e l i m i n a t e d

was

cycle

in Chart

information,

that a Set is on the N - t e r m i n a l remaining

are:

for AM1 are g i v e n

with the AM1

site

the

(data not shown).

65 % of the cofactor

either after amino acid or by sequence

By a n a l o g y binding

is

to

(M+H) + = 388

The amino acid c o m p o s i t i o n

MADH from Meth~lobacterium sp.

of

The

dihydro-form.

sequential

in a c c o r d w i t h two site a t t a c h m e n t

subunit

these

showed the

Analysis.

carboxymethylcysteine;

These d a t a are

= 388.

corresponds

is again the reduced

clearly

Se~ence

The results

;(M-H)-

respectively.

cofactor

analysis.

390

in the analysis,

390 species

ion spectra

and

W3AI

cofactor

certainty. Pro.

than Val,

This

The means

thus the o n l y

i).

50 55 - Leu - Ala - Ser - Set - Set - ~I - Val - Ala - Set i

Cofactor I

- Ile - Ile - X 2 - Cys - Phe - GIy 150 .

B.

.

.

.

.

.

.

.

.

t

- Ser - Pro - Set q Set - X|I - V a l

~ Ala

I

Cofactor

i

J

|

t

!, Val - X 2 - Cys ! Phe

, L

! . . . . .

-

-

I

c~ _ I

Chart I: A. The amino acid sequences in the vicinity of the cofactor binding sites of MADH from Methylobacterium sp. AMI. The numbers are the position in the sequence counting from the N-terminus. B. The suspected amino acid sequences around the cofactor binding sites of MADH from bacterium W3AI. The X's indicate the positions to which the cofactor is attached, and CM = carboxymethyl. The boxed area indicates the components remaining after the aminopepidase-M and carboxypeptidase-Y digestion of the major peptide. 565

Vo1,141, No. 2,1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mass S p e c t r ~ Arkalysis of the Cofactor (M+H) +,

for

several

peptides

[~ptides.

The

parent

ion

masses,

were measured and data were in accord with the

amino acid analyses (data not shown).

The major peptide gave

(M+H) +

ions

of

1431 and 1429, suggesting that thesemicarbazide derivative of the cofactor can be reduced to a dihydro-form as was 2,7,9-tricarboxy-P~-semicarbazone. The daughter spectra for all the peptides, semicarbazone,

as well as 2,7,9-tricarboxy-PQQ-

indicated fragments resulting from the

semicarbazone

side chain.

same

cleavage

of

the

Little more structural information was derived from

the daughter spectra of the peptide samples. The

major

peptide

carboxypeptidase-Y.

was

further

treated

and

fragmentation

aminopeptidase-M

Amino acid analysis indicated that CM-cys,

were still present (part B of Chart i). (M+H) + = 940

with

(M-H)- = 938.

The

and

Set, and 2 Val

The parent ions for this material were daughter

spectra

showed

considerable

which resulted in the identification of the amino acids attached

to the cofator.

The absence of peaks associated with the loss of HCOOH or

CO 2

in the daughter spectra indicate a lack of COOH groups for this cofactor. DISCUSSION

The

mass

spectral

data

confirm

that the structure of the non-covalently

bound cofactor of methanol dehydrogenase and media

of

bacterium

W3AI

that

isolated

is the tricarboxy-l~ I.

this organism is, however, quite different.

from

the

growth

The cofactor of MADH from

Although the tricyclic ~

nucleus

seems to be maintained, no COOH groups are present, and a Cys thioether, -CH 2-

bridge,

and

a

Set

oxygen ether link this quinone to the polypeptide.

Caution must be used in relying

too

fragment

those

system.

ions,

in

particular

8

heavily

on

some

at

the

2,3,7,8,

spectral

data

would produce.

Cys-S-CH2-,

alone.

Fig.

the

less

intense

or

9

position

is

With the assumption that the hydrogens at the 3 and

position of 1 are maintained on the MADH cofactor,

to assign Set-O-,

of

which result from cleavages of the ring

Assignment of the substituents

speculative at this point.

via a

it is still not possible

and H- to the remaining 3 sites from

the

mass

1 shows a possible structure and the fragments it

The sites for substitution of the Cys-S-CH 2- and Set-O- can

566

be

Vol. 1 41, No. 2, 1 986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 0 0 H2N-CH-C-NH-CH-C-NH-CH-C- OH I I I CHz CH CH " "CH3 OH H ~ , , ' t OH, ~N

0 0 0 H2N-CH-C -NH-CH-C-NH-CH-C-OH I I I cH2 cH2 cH OH H ~ N _ ~ Z , , C H~"s "CH3

',~

608

c~ " - ~ ~ ' o CH3/CH63oJ,.zHIS*SHI621 c.

~°L2. ,, NH- C-NH~,

o

Cx.H3/cH3

o

c.o I

n

S

NHt C-NH~.

c..o /

ii

ii

H2N-CH-C-NH~H-C- NH-CH~C-OH 0

"I2N-CH- CtNH ~'~C'~ NH-CH'C-OH 0 ~4ZlH ~ CH'- S- CH2-C- OH

824

I ~, L~S-CH~'-C - OH CH:~L'

8o2

A

B

Figure I: One possible structure of the cofactor of MADH. A presents the positive ion fragments from both the FAB and the daughter spectra. B presents the negative ion fragments from both the FAB and the daughter spectra.

interchanged

or

both can be on the pyridine ring.

the starting material for this cofactor,

If 2,7,9-tricarboxy-PQQ is

2 COOH groups have been lost and

the

3rd has likely been reduced and is the precursor for the -CH 2- bridge to Cys. The mass spectral analysis offered no evidence pertaining to the site of the semicarbazide adduct.

The tentative assignment is at the 5 position since this

carbon is the most electrophilic center for ~ Q

and its analogs (12).

De Beer et al. (13) reported the comparison of the ENDOR spectra of MADH and methanol dehydrogenase.

For the latter enzyme they found hyperfine coupling of

2 protons in the cofactor radical. the unpaired electron. cofactors

for

For MADH they observed 3 protons coupled to

Similar ESR spectra lead to the interpretation that the

both enzymes were the same and that the 3rd proton coupling for

methanol dehydrogenase was masked (13). aromatic

hydrogens

will

couple

to

The current the

data

unpaired

dehydrogenase but three will be for the cofactor in MADH. the

hyperfine

indicate

electron

in

only

two

methanol

Additionally,

2

of

coupling constants for the MADH are very similar to the 2 found

for methanol dehydrogenase

(13).

This suggests that the hydrogens at the 3 and

8 position of the PQQ ring system are conserved in the cofactor from MADH. The structure of the cofactor for MADH has implications for the biosynthesis of PQQ and the mechanism involved in its linkage to the polypeptide. 567

Questions

Vol. 141, No. 2, 1986

arise

as

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to whether 2,7,9-tricarboxy-PQQ preceeds the cofactor linked to MADH

in the biosynthetic pathway or whether point.

Accepted

notions

concerning

the

pathway

structural

information

at

an

earlier

the structure of both non-covalently and

covalently bound FQQ should be re-evaluated in light Direct

diverges

is crucial,

of

the

data

presented.

and less reliance placed on those

methods which measure its biological, spectral, and chromatographic properties. ACKSOWL~D~E~TS This work was supported in part by Program Project grant HL 16251 (Dr. T.P. Singer, principal investigator) from the NIH, and the Veterans Administration. Mass spectra were acquired at the Mass Spectral Facility, Michigan State Univ., which is supported by grant RR 00480 from the Biotechnology Resource Branch, Div. of Research Resources, NIH. One of the authors (W.S.M.) wishes to thank Dr. W.C. Kenney, with whom the foundation for this work was laid. REFERENCES

i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12.

13.

Duine, J.A., Frank, J., and Jongejan, J.A. (1986) FEMS Microbiol. key. 32, 167-178. Ameyama, M., Hayashi, M., Matsushita, K.E., Shinagawa, E., Matsushita, K., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565. Ameyama, M., Shinagawa, E., Matsushita, K., Takimoto, K., Nakashima, K., and Adachi, O. (1985) ibid. 49, 3623-3626. Ameyama, M., Shinagawa, E., Matsushita, K., Nakashima, K., and Adachi, O. (1985) ibid. 49, 699-709. Ameyama, M., and Adachi, O. (1986) Chem. Abst. 104, 205512f. Lobenstein-Verbeek, J.A., Jongejan, J.A., Frank, J., and Duine, J.A. (1984) FEBS Lett. 270, 305-309. Kenney, W.C., and McIntire, W. (1983) Biochemistrv 22, 3858-3868. Ishii, Y., Hase, T., Fukumori, Y., Matsubara, H., and Tobari, J. (1983) J. Biochem. (Tokvo) 93, 107-119. Duine, J.A., and Frank, J. (1980) Biochem. J. 187, 221-226. McIntire, W., Singer, T.P., Smith, A.J., and Mathews, F.S. (1986) Biochemistrg (in press). Gainor, J.A., and Weinreb, S.M. (1982) J. Org. Chem. 47, 2833-2837. Dekker, R.H. ,Duine, J.A., Verveil, P.E.J., and Westerling, J. (1982) Eur. J. Biochem 125, 69-73; Forrest, H.S., Cruse, W.B.T., and Kennard, O. (1979) Nature 280, 843-844; Sleath, P.R., Noar, J.B., Eberlein, G.A., and Bruice, T.C., (1985) J. Amer. Chem. Hoc. 107, 3328-3338. De Beer, R., Duine, J.A., Frank, J., and Large, P.J. (1980) Biochim. Biophgs. Acta 622, 370-374.

568