Báo cáo khoa học: Post-translational modifications in the active site region of methyl-coenzyme M reductase from methanogenic and methanotrophic archaea potx

Surprisingly, in the active site region, five modified amino acids were found: thioglycine a445, forming a thioxo peptide thioamide bond with tyrosine a446, S-methylcyste-ine a452, 2-S-met

of methyl-coenzyme M reductase from methanogenic

and methanotrophic archaea

Jo¨rg Kahnt1, Ba¨rbel Buchenau1, Felix Mahlert1, Martin Kru¨ger2, Seigo Shima1

and Rudolf K Thauer1

1 Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

2 Bundesanstalt fu¨r Geowissenschaften und Rohstoffe, Hannover, Germany

Methane is formed in methanogenic archaea from

methyl-coenzyme M by reduction with coenzyme B

This reaction is catalyzed by methyl-coenzyme M

reductase (MCR) The 300 kDa enzyme is composed

of three different subunits in an a2b2c2 arrangement

and contains 2 mol of the nickel tetrapyrrole

coen-zyme F430, tightly bound The prosthetic group has to

be in the Ni(I) oxidation state for the enzyme to be

active Some methanogenic archaea contain two MCR

isoenzymes, designated MCR I and MCR II, the

syn-thesis of which is differentially regulated [1] There is

circumstantial evidence that MCR is also involved in

the anaerobic oxidation of methane with sulfate by

methanotrophic archaea of the ANME-1, ANME-2 or

ANME-3 clusters [2–4]

The crystal structure of MCR I from

Methanother-mobacter marburgensis has been resolved to 1.16 A˚

[5–8] The structure revealed two identical F430-binding sites, roughly 50 A˚ apart Each F430 is buried deeply within the protein complex and is accessible from the protein surface only via a 50 A˚ long channel, which at its narrowest part is only 6 A˚ in diameter The channel and the coenzyme-binding sites are formed mainly by hydrophobic residues of subunits a, a¢, b and c, and a¢, a, b¢ and c¢, respectively (the prime superscript indi-cates the second identical subunit) Surprisingly, in the active site region, five modified amino acids were found: thioglycine a445, forming a thioxo peptide (thioamide) bond with tyrosine a446, S-methylcyste-ine a452, 2-(S)-methylglutamS-methylcyste-ine a400, 1-N-methylhisti-dine a257 (3-methylhistidine according to IUPAC nomenclature) and 5-(S)-methylarginine a271 (Fig 1) The modifications are introduced after translation, as the DNA sequence of the encoding mcrA gene shows

Keywords

methanogenic archaea; methanotrophic

archaea; methylated amino acids;

methyl-coenzyme M reductase; thioxo peptides

Correspondence

R Thauer, Max-Planck-Institute fu¨r

terrestrische Mikrobiologie,

Karl-von-Frisch-Strasse, D-35043 Marburg, Germany

Fax: +49 6421 178109

Tel: +49 6421 178101

E-mail: thauer@mpi-marburg.mpg.de

Website: http://www.mpi-marburg.mpg.de/

(Received 11 June 2007, revised 23 July

2007, accepted 26 July 2007)

doi:10.1111/j.1742-4658.2007.06016.x

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step

in methanogenic archaea Isoenzyme I from Methanothermobacter marbur-gensis

2 was shown to contain a thioxo peptide bond and four methylated amino acids in the active site region We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post-translational methylations The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophi-lus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri Two MCRs isolated from Black Sea mats contain-ing mainly methanotrophic archaea of the ANME-1 cluster were also ana-lyzed

Abbreviation

MCR, methyl-coenzyme M reductase.

no unusual codons or unusual codon usages at the

positions in which the five modified amino acids were

found Via in vivo labeling experiments with

l-(methyl-D3)-methionine, it was shown that the methyl groups

in the four methylated amino acids are introduced

cotranslationally or post-translationally by specific

S-adenosylmethionine-dependent protein methylases

[9] How the sulfur is transferred into the carboxamide

group of glycine in the peptide chain remains to be

shown

Neither the functions of the five modifications nor

whether the modifications are present in MCRs from

all methanogens

structures deduced from the DNA sequences reveals

that the five amino acid positions are conserved in

MCRs from all methanogenic archaea [9] However, in

the gene for the a-subunit of MCR from

methano-trophic archaea of the ANME-1 cluster, there is a

codon for a valine, whereas in mcrA from

methano-genic archaea, there is a codon for a glutamine [2]

Methanogenic archaea and methanotrophic archaea all belong to the kingdom of Euryarchaeota They are classified on the basis of their 16S RNA sequence in five orders, Methanobacteriales, Methanopyrales, Met-hanococcales, Methanomicrobiales and Methanosarci-nales [10] The phylogenetic distance between the archaeal orders is as large as between, for example, proteobacteria and Gram-positive bacteria The phy-logeny is reflected in the primary structure of the MCR a-subunit, which can therefore be used to clas-sify methanogens [11]

In the work reported here, we have analyzed by MS the MCR from at least one representative of each of the five orders of methanogenic archaea and from two methanotrophic archaea of the ANME-1 cluster

We have included in the analysis both MCR I and MCR II from Methanothermobacter marburgensis (growth temperature optimum 65C) and MCR I from a mesophilic (37C) and a hyperthermophilic (85C) Methanococcus species The analysis revealed that the thioxo peptide bond is conserved in all MCRs investigated, but that there are differences in the post-translational methylations Specifically, Cys a452 is not methylated in the enzyme from the hyperthermophilic Methanocaldococcus jannaschii and Methanopyrus kandleri, and Gln a400 is not methylated in Methano-sarcina barkeri

Results

Up to now, the crystal structures of three methyl-coen-zyme M reductases have been resolved, MCR isoen-zyme I from Methanothermobacter marburgensis, MCR from Methanosarcina barkeri, and MCR from Methano-pyrus kandleri[7] In case of the enzyme from Methano-thermobacter marburgensis and Methanosarcina barkeri, the resolution was high enough to identify the post-translational modifications; in the case of the Methanopyrus kandlerienzyme, it was not Attempts to obtain diffracting crystals of MCR II from Methano-thermobacter marburgensis and of the MCR from the other methanogens mentioned below were not success-ful We therefore searched for post-translational modifications in the active site region by subjecting the a-subunit of MCRs to tryptic digestion, followed by separation and sequencing of the peptides of interest Either before or after the tryptic digestion, any oxidized cysteine residues were reduced with dithiothreitol and subsequently alkylated with 4-vinylpyridine

Either of two methods for sequencing the alkylated tryptic peptides were employed: (a) the tryptic peptide was subjected to partial hydrolysis with aminopepti-dase M and the partial hydrolysate was then analyzed

O -O

NH3+

NH+

N

H 3 C

H 3 C

S O

-O

NH3+

H2N N O

NH3+

CH 3

H

H2N O

NH3+

H 3 C

N

S

H

N 1 -Methylhistidine

S-Methylcysteine

5-(S)-Methylarginine

2-(S)-Methylglutamine

Thiopeptide bound thioglycine

Fig 1 Post-translationally modified amino acids in the a-subunit of

methyl-coenzyme M reductase isoenzyme I from

Methanothermo-bacter marburgensis Two of these modified amino acids,

2-(S)-methylgutamine and 5-(S)-methylarginine, have until now not been

found in any other protein 1-N-Methylhistidine ¼ 3-methylhistidine

according to IUPAC nomenclature.

by MALDI-TOF MS (Fig 2); or (b) the tryptic

pep-tide was subjected to MALDI-TOF MS⁄ MS (Fig 3)

As a control, the five modifications within the active

site region of MCR I from Methanothermobacter

mar-burgensiswere found using both methods Where

indi-cated, proteases other than trypsin were used for

peptide generation

Tryptic peptide containing thioglycine and

S-methylcysteine or cysteine

In the tryptic digest of the MCR a-subunit from all

methanogens investigated, a peptide containing a

thi-oxo peptide was found (Table 1) The sequence

LGFY-thioxoglycine-YDLQDQC is highly conserved

in the nine tryptic peptides analyzed Very few

varia-tions were found, namely a phenylalanine instead of a

tyrosine directly before the thioglycine (Methanoculleus

thermophilus and Methanosarcina barkeri) or directly

after the thioglycine (Methanosarcina barkeri), and an

alanine instead of an aspartate two positions from the

thioglycine (Methanopyrus kandleri) The cysteine

fol-lowing the conserved glutamine (Q) is methylated in

the two isoenzymes from Methanothermobacter

mar-burgensisand in the enzymes from Methanococcus

vol-tae, Methanoculleus thermophilus and Methanosarcina barkeri, but is not methylated in the enzyme from Met-hanopyrus kandleriand Methanocaldococcus jannaschii The thiocarbonyl group in thiopeptides can undergo

a slow reaction with water, in which the 32-sulfur is replaced by a 16-oxygen This explains why, in some spectra, a parallel sequence shifted by 16 Da to smaller masses was seen (Fig 2)

In two MCRs from methanotrophic archaea of the ANME-1 cluster [11,12] a tryptic peptide with the N-terminal sequence LGFYGYDL QDQCTAC

found (amino acids modified in other MCR are in bold type), but the glycine was not thioxylated and the cysteine was not methylated (Table 1) As the MCR was isolated from microbial mats from the Black Sea and could not be tested for activity, the possibility can-not be excluded that the enzyme once had a thioxo group but lost it by spontaneous hydrolysis

Peptides containing N-methylhistidine, 5-methyl-arginine or 2-methylglutamine

The methylated histidine (H257) was found in the a-sub-unit of MCRs from all methanogenic archaea and of the two MCRs from the methanotrophic archaea of the

1325.5

1453.61

1566.74 1637.8

1800.92

2037.05

Thio

1873.94

*

*

*

Mass (m/z)

0

100

2184.15

1210.42

Fig 2 MALDI-TOF MS of the peptide mixture generated via aminopeptidase M from the thioglycine-containing tryptic peptide of the MCR a-subunit from Methanopyrus kandleri The purified enzyme was denatured in SDS, digested with trypsin, and then alkylated with 4-vinylpyri-dine Subsequently, the tryptic peptides were separated by HPLC and analyzed by MALDI-TOF MS The peptide with a mass of 2353.28 Da (predicted to contain the thioglycine) was partially hydrolyzed by aminopeptidase M, and the partial hydrolysate was analyzed by MALDI-TOF

MS From the peptide ladder, the N-terminal amino acid sequence was found to be LGFY-thioglycine-YALQD The mass peaks labeled with

an asterisk have a mass that is 16 Da smaller than those of the respective thioglycine-containing peptides The thiocarbonyl group in thiopep-tides can undergo a slow reaction with water in which the 32-sulfur is replaced by an 16-oxygen ThioGly, thioglycine.

ANME-1 cluster (Table 2) The sequence around the

methylated histidine is shown in Fig 4

The methylated arginine (R271) is also highly

con-served in the a-subunit of MCRs from methanogenic

archaea However, in the MCR from the

methano-trophic archaea of the ANME-1 cluster, the arginine

in the respective tryptic peptide was not methylated (Table 2)

The glutamine (Q400) is not methylated in the a-subunit of MCR from Methanosarcina barkeri In

Table 1.

10;11 Amino acid sequence of the MCR a-subunit tryptic peptide containing the thioglycine G*, thioglycine; C, S-methylcysteine Amino acids modified in most MCR are indicated by bold type.

MCR from

Sequence of the thioglycine-containing tryptic peptide

Methanothermobacter

marburgensis (MCR I)

LGFYG*YDLQDQC*GASNVFSIRa,b Methanothermobacter

marburgensis (MCR II)

LGFYG*YDLQDQC*GASNSLSIRa Methanopyrus

kandleri

LGFYG*YALQDQCGAANSLSVRa

Methanocaldococcus

jannaschii (MCR I)

LGFYG*YDLQDQCGAANSLSFRb Methanococcus

voltae

LGFYG*YDLQDQC*GASNSLAIRa,c

Methanoculleus

thermophilus (MCR I)

LGFFG*YDLQDQC*GSANSLSIRc Methanosarcina

barkeri

LGFFG*FDLQDQC*GATNVLSYQGDEGLPDELRc Methanotrophic archaea

of the ANME-1b cluster

LGFYGYDLQDQCTACGSYSYQSDEGMPFEMRc,d

a Sequence determined via aminopeptidase M and MALDI-TOF MS b Sequence taken from Selmer et al [9] c Sequence determined via MALDI-TOF MS ⁄ MS d Sequence of peptide obtained from two different MCRs isolated from the Black Sea mats.

Mass (m/z) 0

1257.15

Pyridyl-ethyl Cys

321.43

608.27

521.31 408.34

722.27 793.26 864.25

921.23

1964.0 1891.63 1728.36 1613.4

1500.38 1372.26 1129.15

174.59

Thio Gly

2127.99

2446.36 100

Fig 3 MALDI-TOF MS ⁄ MS of the thioglycine-containing tryptic peptide of the MCR a-subunit from Methanocaldococcus jannaschii The purified enzyme was denatured in SDS, alkylated with 4-vinylpyridine, and then subjected to SDS ⁄ PAGE After in-gel digestion of the a-sub-unit with trypsin, the generated peptides were analyzed by MALDI-TOF MS The peptide with a mass of 2446.36 Da (predicted to contain the thioglycine) was then analyzed by MALDI-TOF MS ⁄ MS From the fragment pattern, the following amino acid sequence is deduced: LGFY-thioglycine-YDLQDQ-pyridylethylCys-GAANSLSFR ThioGly, thioglycine.

the two MCRs from the ANME-1 cluster, there is a

valine instead of a glutamine (Table 2)

Discussion

Methanogenic archaea are dependent on methane

formation for growth, and thus on a functional

methyl-coenzyme M reductase [1,13] Therefore, a genetic

analysis of methanogenic archaea with respect to the

function of the post-transcriptional modifications of

MCR is presently not possible [14,15] A reversed

genetic approach is also not in sight, because the genes

required for the biosynthesis of the nickel cofactor F430

[16] and for the post-translational modifications [9] are

not yet known [17,18] Also, the in vitro reconstitution

of active enzyme from its subunits and cofactor has

proven very difficult; very little activity is recovered

[19] Therefore, the function of the post-translational

modifications can at present only be approached by

comparison of MCRs from archaea differing in

phylo-genetic relationship and⁄ or growth conditions

An early hypothesis was that the post-translational

or cotranslational modifications modulate the kinetic

properties (Km and Vmax) of MCR, which is why we

started the analysis with isoenzyme II from

Methano-thermobacter marburgensis, which differs considerably

in Km and Vmax from isoenzyme I [20] However, this

hypothesis had to be abandoned when we found that

the two isoenzymes were identical with respect to their

post-translational modifications (Table 2)

His a257 is methylated in all analyzed MCRs, indi-cating an essential function (Table 2) The residue 1-N-methylhistidine is part of the coenzyme B-binding site Its unmethylated nitrogen atom Ne2 donates the shortest of the three hydrogen bonds of the protein to the phosphate group of coenzyme B The distance of 2.65 A˚ indicates the presence of a strong hydrogen bond with fully overlapping orbitals Owing to the positive inductive effect, the pKa of the methylated amino acid is expected to increase slightly (imidazole and N-methylimidazole differ in their pKa by 0.1), and this will affect the strength of coenzyme B binding A more important function of the methyl group may involved improved orientation of the histidine inside the coenzyme B-binding site With the methylation, the histidine residue is prevented from forming two alter-nate nitrogen positions by rotating around the angle

v2, due to occlusion by a peptide oxygen from the pre-ceding residue [21]

The functions of the three other methylations are less obvious, in part because they are not conserved in all MCR enzymes (Table 2) The reduction of methyl-coenzyme M with methyl-coenzyme B takes place in a hydro-phobic pocket in the complete absence of water It involves radical intermediates that are very reactive [5,18] This is probably why the active site of this enzyme is lined up primarily with unreactive aromatic and aliphatic amino acid residues The methyl groups

of S-methylcysteine, 5-methylarginine and 2-methylglu-tamine are oriented towards the active site chamber

Table 2 Amino acid modifications found in the a-subunit of MCRs from methanogenic archaea by MS analysis of tryptic peptides Where indicated, the a-subunit was subjected to proteolysis by chymotrypsin, endoproteinase AspN or BrCN rather than by trypsin The data for MCR I from Methanothermobacter marburgensis were taken from Selmer et al [9] NF, respective peptide not found.

Methanothermobacter

marburgensis (MCR I)

Methanothermobacter

marburgensis) (MCR II)

Methanopyrus

kandleri

Methanocaldococcus

jannaschii (MCR I)

Methanococcus

voltae

Methanoculleus

thermophilus (MCR I)

Methanosarcina

barkeri

Methanotrophic archaea

of the ANME-1 cluster

(two MCRs)

a a-Subunit cleaved with chymotrypsin b a-Subunit cleaved with BrCN c a-Subunit digested with AspN.

and have no contact with solvent The function of the

methylations could thus be to protect the respective

amino acid residues from reaction with the radical

intermediates As the methylations provide the amino

acids with additional hydrophobic interactions,

another function could be to restrict the

conforma-tional flexibility of the residues, as has been discussed

for post-transcriptional tRNA modifications [22–24]

The observed differences in methylation are difficult

to explain, because they may be compensated for by

differences in positioning or properties of nearby

resi-dues, which are not directly evident

A function for the thioxo peptide bond (for

proper-ties see Pfeifer et al [25] and Artis & Lipton [26]) was

proposed in the catalytic cycle of MCR In the final

step of the cycle, a disulfide anion radical of

coen-zyme M and coencoen-zyme B is formed, which is oxidized

to the heterodisulfide by reducing the active site’s nickel back to the Ni(I) oxidation state [5,18] The thi-oxo group is positioned such that it could be involved

in electron transport from the disulfide anion radical

to the nickel The redox potential of the thioxo pep-tide⁄ radical couple is estimated to lie between that of the disulfide⁄ disulfide anion radical couple () 1.7 V) [27] and the F430[Ni(II)]⁄ F430[Ni(I)] couple () 0.64 V) [18] It has also been suggested that upon a one-elec-tron reduction of the thioxo peptide bond, a change from the trans to the cis conformation (similar to the light-induced switch of the thioxo peptide bond from trans to cis [28,29]) could occur, and that this confor-mational change could be involved in coupling the two active sites [4,30,31] The finding of the thioxo peptide

Fig 4 Partial amino acid sequence of the a-subunit of MCRs from six methanogenic archaea of five different orders and from two methano-trophic archaea of the ANME-1 cluster The numbering is that for the a-subunit of MCR I from Methanothermobacter marburgensis The amino acids, which in MCR from Methanothermobacter marburgensis are post-translationally modified, are highlighted in bold The ANME-1a sequence (2216) was provided by A Meyerdierks, Max Planck Institute for Marine Microbiology in Bremen, and the ANME-1b sequence was taken from Lyoyd et al [40].

bond in MCRs from all methanogenic archaea

sup-ports this hypothesis, whereas the finding that the two

MCRs isolated from the Black Sea microbial mats

lacks this bond does not As the thioxo peptide bond

can lose its sulfur by hydrolysis, and as the MCR

extracted from the microbial mats from the Black Sea

was inactive, the function of the thioxo peptide bond

as proposed above cannot yet be dismissed This

ques-tion will remain open until methanotrophic archaea

can be grown in the laboratory and until active MCRs

can be isolated from them for analysis of the presence

of the thioxo peptide bond

Methylated amino acids are found in many proteins,

although the presence of 5-methylarginine and

2-meth-ylglutamine has, until now, only been reported for

MCR [9] A thioxo peptide bond in a protein has not

been encountered before However, such a bond has

been found, for example, in methanobactin from

meth-ane-oxidizing bacteria [32] and thioviridamide from

Streptomyces olivoviridis [33] How these

nonriboso-mally synthesized polypeptides are thioxylated is not

known A mechanism similar to that described for the

sulfuration of the C-terminal glycine in ThiS and

MoaD, involved in thiamine biosynthesis and

molyb-dopterin biosynthesis, respectively, and for the

sulfura-tion of uridine in tRNA to thiouridine can be

envisaged [34–36] This mechanism probably also

applies for the synthesis of

pyridine-2,6-bis(thiocarb-oxylate) from dipicolinic acid in Pseudomonas stutzeri

[37–39]

Experimental procedures

Methanothermobacter marburgensis (DSMZ 2133) (growth

temperature optimum 65C), Methanopyrus kandleri

(DSMZ 6324) (98C), Methanococcus voltae (DSMZ 1537)

(37C), Methanocaldococcus jannaschii (DSMZ 2661)

(85C), Methanoculleus thermophilus (DSMZ 3915) (57 C)

and Methanosarcina barkeri (strain Fusaro) (DSMZ 804)

(38C) were obtained from the Deutsche Sammlung von

Mikroorganismen und Zellkulturen (Braunschweig,

Ger-many) The first three methanogenic archaea were grown

on H2 and CO2, Methanoculleus thermophilus on

isopropa-nol, H2and CO2, and Methanosarcina barkeri on methanol

The microbial mats containing methanotrophic archaea of

the ANME-1 cluster were from the Black Sea [2] MCR

was purified from cells by published procedures [1]

Of the methanogenic archaea mentioned above,

Methano-thermobacter marburgensis, Methanocaldococcus jannaschii

and Methanoculleus thermophilus contain two MCR

iso-enzymes, whereas Methanococcus voltae, Methanopyrus

kandleri and Methanosarcina barkeri do not have MCR

isoenzymes

For determination of the amino acid modifications in the a-subunit of MCR, the a-subunit was subjected to proteo-lysis by trypsin, and then the tryptic peptides, after separa-tion, were analyzed by MALDI-TOF MS Where indicted, the a-subunit was subjected to proteolysis by chymotrypsin (sequencing grade; Roche, Mannheim, Germany), endopro-teinase AspN (sequencing grade; Roche) or BrCN rather than by trypsin

MALDI-TOF MS analysis of tryptic peptides after partial digestion with aminopeptidase M

Purified MCR (2 mg of protein in 25 lL) was supplemented with SDS and dithiothreitol to final concentrations of 1.5% and 200 mm, respectively, and then incubated for 10 min at

95C to fully denature the protein Subsequently, the solu-tion was diluted 10-fold with 100 mm NH4HCO3(pH 8.3), supplemented with 0.2 mg of trypsin (diphenyl carbamyl chloride

6 -treated, 11392 U mg)1; Fluka, Buchs, Switzerland), and incubated for 12 h at 37C Then, excess 4-vinylpyri-dine (60 mm) was added to alkylate the cysteine-derived thiol groups After incubation for 30 min at 70C, the non-reacted vinylpyridine was removed by evacuation to dryness

in a Savant-SpeedVac SC110; (Life Sciences International, Frankfurt, Germany)

7 and the dried material containing the tryptic peptides was redissolved in 400 lL of H2O After removal of SDS using a Detergent-Out column (Genotech,

St Louis, MO, USA), the peptides were separated by RP-HPLC and analyzed by MALDI-TOF MS The peptide whose molecular mass matched with that predicted for the modified amino acid containing tryptic peptide was partially hydrolyzed with 10 mU of aminopeptidase M (Roche) at

pH 7.1 for 30 min at room temperature, and the partial hydrolysate was then analyzed via MALDI-TOF MS (Voyager DE-RP; ABI, Darmstadt, Germany; 337 nm N2 laser) From the peptide ladder, the N-terminal amino acid sequence of the peptides was obtained as shown in Fig 2 for the thioglycine-containing tryptic peptide of the MCR a-subunit from Methanopyrus kandleri

MALDI-TOF MS⁄ MS analysis of tryptic peptides Purified MCR (0.15 mg) was denatured in SDS (0.15%) containing dithiothreitol (6 mm), alkylated with excess 4-vi-nylpyridine (30 mm) as described above, and then subjected

to SDS⁄ PAGE followed by staining with Coomassie Blue The band corresponding to the a-subunit was cut out, di-stained and dried under vacuum, and the dried material was suspended in 30 lL of 10 mm NH4HCO3(pH 8.3) con-taining 0.6 lg of trypsin (sequence grade, modified; Pro-mega, Madison, WI, USA) and 10% acetonitrile After incubation at room temperature for 12 h, the trypsin digest was extracted, and the extract was applied to a C18 Pep-Map 100 nano LC column

the separation of the peptides, which were subsequently

analyzed by MALDI-TOF MS The peptide whose mass

matched that predicted for the modified amino acid

con-taining tryptic peptide was analyzed by MALDI-TOF

MS⁄ MS (Ultraflex; Bruker, Bremen, Germany) From the

fragment pattern, the amino acid sequence was obtained as

exemplified in Fig 3 for the thioglycine-containing tryptic

peptide of the MCR a-subunit from Methanocaldococcus

jannaschii

Acknowledgements

This work was supported by the Max Planck Society

and the Fonds der Chemischen Industrie

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