©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Sequencing, and Transcriptional Analysis of the Coenzyme F-dependent Methylene-5,6,7,8-tetrahydromethanopterin Dehydrogenase Gene from Methanobacterium thermoautotrophicum Strain Marburg and Functional Expression in Escherichia coli(*)

(Received for publication, September 27, 1994)

Biswarup Mukhopadhyay (§) Endang Purwantini Todd D. Pihl (1) John N. Reeve (1) Lacy Daniels (¶)

From the Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Department of Microbiology, Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two methylenetetrahydromethanopterin dehydrogenases have been purified from Methanobacterium thermoautotrophicum strain Marburg: one (MTD) is coenzyme F-dependent and oxygen-stable (Mukhopadhyay, B., and Daniels, L.(1989) Can. J. Microbiol. 35, 499-507), and the other (MTH) is coenzyme F-independent (or hydrogenase-type) and oxygen-sensitive (Zirngibl, C., Hedderich, R., and Thauer, R. K.(1990) FEBS Lett. 261, 112-116). Based on the NH (2)-terminal sequence of MTD, a 36-mer oligonucleotide was designed and used to identify and clone a 6.1-kilobase pair EcoRI fragment of M. thermoautotrophicum DNA. Sequencing of this fragment revealed an 825-base pair (bp) MTD encoding gene (mtd), which was expressed in Escherichia coli yielding an enzyme that, like the native enzyme, was oxygen-stable, strictly dependent on coenzyme F, thermostable, thermophilic, and exhibited maximum activity at an acidic pH. The amino acid sequence predicts that MTD is a hydrophobic and acidic protein with no identifiable homology to MTH (von Bunau, R., Zirngibl, C., Thauer, R. K., and Klein, A.(1991) Eur. J. Biochem. 202, 1205-1208), but comparisons with coenzyme F utilizing enzymes revealed a conserved region at the NH(2) terminus of MTD that could correspond to the ability to interact with coenzyme F. The mtd transcript was 900 nucleotides long and initiated 8 bp upstream of the translation initiation codon and 22 bp downstream from an archaeal promoter sequence. The mtd coding sequence was followed by several poly(dT) sequences and an inverted repeat that could be transcription termination signals.


INTRODUCTION

Methanogens are strictly anaerobic archaea and they reduce CO(2) to methane using the following pathway(1) : CO(2) formyl-MF (^1) N^5-formyl-H(4)MPT N^5,N-methenyl-H(4)MPT N^5,N-methylene-H(4)MPT N^5-methyl-H(4)MPT CH(3)-S-CoM CH(4), where methanofuran (MF), tetrahydromethanopterin (H(4)MPT), and coenzyme M (HS-CoM) are C(1)-carrying cofactors(1) .

The interconversion of methylene-H(4)MPT (H(2)C=H(4)MPT) and methenyl-H(4)MPT (HC=H(4)MPT) is catalyzed by H(2)C=H(4)MPT dehydrogenase. Cell extracts of Methanobacterium thermoautotrophicum strain Marburg (M. thermoautotrophicum Marburg) exhibit two types of H(2)C=H(4)MPT dehydrogenase activity; one is air-stable and F-dependent(2) , and the other is air-sensitive and F-independent(2, 3) . We purified the F-dependent enzyme, a multimer of 32-kDa subunits(2, 4) , and Zirngibl et al.(3) purified the F-independent (hydrogenase-type) enzyme, a 43-kDa single-polypeptide protein. Initially it was thought that the ``methanobacterium-type'' methanogens (those possessing pseudomureins in their cell walls) contained only the hydrogenase-type enzyme (5) and the F-dependent activity in the cell extract of M. thermoautotrophicum Marburg might result from an in vitro processing of the hydrogenase-type enzyme. When von Bunau et al.(6) cloned and sequenced the gene encoding the hydrogenase-type enzyme, they discovered that this enzyme was not predicted to harbor the NH(2)-terminal amino acid sequence established for the F-dependent enzyme. Here we report the cloning, functional expression in Escherichia coli, sequencing, and transcriptional analysis of the gene for the F-dependent enzyme and demonstrate conclusively that this enzyme is not a derivative of the F-independent enzyme.


MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Culture Conditions

For cloning, sequencing, and expression of the cloned gene E. coli, XL-1 Blue and pBluescript II SK (Stratagene, La Jolla, CA) were used as the recombinant host and the vector, respectively. E. coli strains were grown in Luria-Bertani medium(7) . M. thermoautotrophicum Marburg was grown on H(2) + CO(2) as described previously (8) except the media contained 10 mM K(2)HPO(4), 15 mM KH(2)PO(4), 0.1 mM trisodium nitrilotriacetate, and 50 µM FeCl(3)bullet6H(2)O(4) . Cells used for RNA isolations were grown in a 2-liter fermentor as described previously(9) .

Purification of Enzymes and Coenzymes, and Assays

Coenzyme F, H(4)MPT, and F-dependent N^5,N-methylene-H(4)MPT dehydrogenase were purified from M. thermoautotrophicum Marburg as described previously(2) . Aerobic and anaerobic cell extracts were prepared in 20 mM potassium phosphate buffer at pH 7(2) . Dehydrogenase was assayed as described previously(2) . For determining pH optima, buffers containing 100 mM of each of MES, Tris, and glacial acetic acid and adjusted to the desired pH with either HCl or NaOH were used; the values calculated (10) for their ionic strengths were between 0.15 and 0.2 unit. For all other assays, 100 mM sodium acetate-acetic acid buffers were used. For thermal stability studies, incubation tubes containing diluted cell extracts were prepared on ice, made anaerobic, placed under argon (1.14 atm)(2) , and then incubated at the test temperatures. The contents of these tubes were assayed for F-dependent dehydrogenase activity before and after incubation. All dehydrogenase assays, except those for determining temperature optima, were carried out at 40 °C. Reaction rates were calculated using the extinction coefficients (for F, H(2)F, and HC=H(4)MPT when monitoring assays at 340 nm and for F when monitoring at 400 nm) determined at the assay pH and temperature(11) . Protein was determined according to Bradford (12) , using the Bio-Rad dye reagent; bovine serum albumin was used as the standard.

Determination of the NH(2)-terminal Sequence of F-dependent Methylene-H(4)MPT Dehydrogenase

Purified F-dependent H(2)C= H(4)MPT dehydrogenase from M. thermoautotrophicum Marburg was electrophoresed under denaturing conditions (13) and electroblotted onto a ProBlott® membrane (Applied Biosystems, Foster City, CA) according to manufacturer's protocol. The dehydrogenase band at the 32-kDa position (1.9 µg or 60 pmol of protein) was excised from the Coomassie Blue (R-250)-stained and dried membrane and used for sequencing by Edman degradation at the Northwestern University Biotechnology Research Service Facility (Evanston, IL) using a model 477A gas phase sequencer with an on-line phenylthiohydantoin analyzer (Applied Biosystems, Foster City, CA).

Antiserum and Immunoblot Analysis

Purified F-dependent H(2)C=H(4)MPT dehydrogenase from M. thermoautotrophicum Marburg (in 20 mM potassium phosphate buffer at pH 7, 20% glycerol, and 300 mM NaCl) was emulsified with an equal volume of Freund's Complete Adjuvant (Difco), and 0.25 ml of this emulsion (4.75 µg of dehydrogenase protein) was injected subcutaneously into a male New Zealand White rabbit; a similar injection was made after 2 days. Further injections were intramuscular and given twice per week. One month after the first inoculation, the rabbit was exsanguinated and serum was prepared(14) .

Western blotting was carried out essentially according to Towbin et al.(15) . Blocking was performed using 0.5% nonfat dry milk. Primary antibody (anti-dehydrogenase antiserum) was used at 1:1000 dilution. The secondary antibody was alkaline phosphatase-conjugated anti-rabbit goat IgG (whole molecule) (Sigma). Immunoreactive bands were detected by using the colorimetric substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma).

Isolation of M. thermoautotrophicum DNA

Genomic DNA of M. thermoautotrophicum Marburg was isolated according to Meakin et al.(16) , but with modifications. A suspension of 2.3 g of cells in 20 ml of 50 mM ammonium bicarbonate (pH 8) containing 50 mM EDTA was incubated, in sequence, with 12 mg of heat-treated Pronase for 1 h at 37 °C, proteinase K (3 mg), and sodium dodecyl sulfate (final concentration, 1%) for 1 h at 65 °C, and 4 mg of dithiothreitol for 30 min at 26 °C. From the resulting lysate, DNA was purified by using a standard technique(7) , except NaCl was added to a final concentration of 500 mM before the ethanol precipitation step.

DNA Techniques

Plasmids were purified from E. coli lysates by using Qiagen tips (Qiagen Inc., Chatsworth, CA). Following restriction enzyme digestion and electrophoresis, the DNA fragments of interest were purified from low melting agarose gels by digesting excised gel slices with beta-agarase (New England Biolabs, Inc., Beverly, MA) followed by ethanol precipitation of the DNA(7) . Cloning of DNA, transformations, and Southern and colony hybridizations were done as described(7) . Oligonucleotide probes were labeled with digoxigenin-ddUTP using the Genius® kit (Boehringer Mannheim) (7) . Prehybridization and hybridization were done at 50 °C, and the post-hybridization wash was at 24 °C. Hybridizing bands or colonies were detected by using alkaline phosphatase-conjugated anti-digoxigenin antibody and the colorimetric substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Sequencing was performed at the University of Iowa DNA Facility using an Applied Biosystems model 373A DNA sequencer with SK and KS primers (Stratagene) and primers based on the accumulated sequences.

RNA Isolation, Primer Extension, and Northern Blot Analysis

Total M. thermoautotrophicum Marburg RNA was isolated following the protocol of Pihl et al.(9) . Primer extension studies and Northern blot experiments were carried out according to Montzka and Steitz (17) and Hennigan and Reeve(18) , respectively.


RESULTS

Cloning and Sequencing of the F-dependent Methylene-H(4)MPT Dehydrogenase Gene (mtd)

The NH(2)-terminal amino acid sequence determined for the F-dependent H(2)C=H(4)MPT dehydrogenase from M. thermoautotrophicum Marburg was VVKIGIIK*GNIGTSPV (*, no phenylthiohydantoin derivative detected), which was identical to the sequence reported by Enßle et al.(19) . Based on this sequence from position 3 to 14 (assuming the unidentified 9th residue as C), the degenerate oligonucleotide 5`-NGTNCC(A/G/T)AT(A/G)TTNCC(G/A)CA(C/T)TT(A/G/T)AT(A/G/T)ATNCC(A/G/T)AT(C/T)TT-3` was synthesized and used to probe an EcoRI digest of M. thermoautotrophicum Marburg genomic DNA. Southern blot analyses revealed a strong hybridization signal corresponding to 6.1-kb EcoRI fragments and three weak signals corresponding to 1-, 5-, and 9.4-kb EcoRI fragments. Four limited libraries comprising the 1-, 5-, 6.1-, and 9.4-kb EcoRI fragments of M. thermoautotrophicum Marburg genomic DNA were constructed in E. coli XL-1 Blue. By screening these libraries by colony hybridization, we identified a positive colony and the recombinant plasmid (designated pPM58) from this clone contained a 6.1-kb DNA insert (Fig. 1A). Further mapping revealed that the probe hybridized to a 1-kb PstI fragment which was subcloned to obtain pBE103 (Fig. 1A).



Figure 1: Restriction map and nucleotide sequence of F-dependent methylene-H(4)MPT dehydrogenase (mtd) clone from M. thermoautotrophicum Marburg. A, restriction map of the 6.1-kb EcoRI insert of plasmid pPM58 and the 1-kb PstI insert of subclone pBE103. The locations and orientations of the mtd gene and the ORFX are shown. B, nucleotide sequence of the mtd gene and flanking regions. The nucleotide sequence is numbered from the first nucleotide of the translation initiating codon of the mtd gene. The deduced amino acid sequences are shown above the nucleotide sequence in single-letter code. A potential ribosome binding sequence for mtd is shown by asterisks. The start sites for the major and minor mtd transcripts, as identified by the primer extension analysis (Fig. 4), are shown by a large and a small arrow, respectively. The putative promoter sequence for mtd is overlined. The inverted repeat sequence indicated by converging arrows and the underlined stretches of T residues downstream of the mtd coding sequence are putative transcription termination signals. The NH(2)terminal amino acid sequence that was used to design the degenerate oligonucleotide probe is shown in italics and is underlined. The vertical arrows correspond to the termini of the cloned PstI fragment in pBE103.




Figure 4: Primer extension analysis. Primer extension reactions were carried out for mtd and ORFX transcripts using the primers listed in the legend to Fig. 3, and 20-µg aliquots of total RNA isolated from cells harvested at a culture OD of 0.4. The products of the primer extension reactions with (+) or without(-) avian myeloblastosis virus reverse transcriptase are shown. The sequencing ladders show sequences of pPM58 DNA obtained with the same primers used in the primer extension reactions. The transcription start sites identified are indicated on the sequence with the largerarrow identifying the major site of transcription initiation and the smallerarrow for the minor start site. Only the results for mtd transcripts are shown.




Figure 3: Northern blot of M. thermoautotrophicum Marburg RNA. Lanes 1 and 2 contained RNA (10 µg in each) isolated from cells harvested at culture OD of 0.4 and 0.9, respectively. The blot was probed with a mtd-specific primer 5`-GTAACAGGTCCAGCACAGG-3` (complimentary to positions 49-67 in Fig. 1B). An identical blot probed with an ORFX-specific primer 5`-TATAACCTCATCACCCAG-3` (complementary to positions -570 to -553) failed to detect an ORFX transcript (data not shown).



The DNA sequence was determined for the cloned F-dependent methylene-H(4)MPT dehydrogenase gene (mtd, methylene-tetrahydromethanopterin dehydrogenase) and for the flanking regions. These sequences, together with the deduced amino acid sequence of the F-dependent dehydrogenase, are given in Fig. 1B. The insert in pBE103 carried a part of an upstream open reading frame (ORFX), the intergenic region, and 86% of the mtd coding sequence (Fig. 1, A and B).

Sequence Analysis

The mtd gene was 825 bp in length with ATG and TAG as initiation and termination codons, respectively. The sequence (5`-TGGTGATC-3`) located 2 bp upstream of the ATG codon is complementary to the sequence at the 3`-end of M. thermoautotrophicum Marburg 16 S rRNA (20) and could therefore function as a ribosome binding site. Upstream of mtd and separated from it by a 168-bp AT-rich intergenic region is a 444-bp open reading frame (ORFX; Fig. 1, A and B). Downstream of mtd are oligo(dT) sequences beginning at positions 844, 879, 914, 1091, and 1143, and a short inverted repeat located between positions 853 and 864. The mtd sequence is 50 mol % G + C consistent with the overall 48 mol % G + C content of the M. thermoautotrophicum Marburg genome(21) . The mtd gene followed the pattern of codon usage seen with the other M. thermoautotrophicum genes (6, 22, 23, 24, 25, 26, 27, 28) , except U was the most often found base in third positions of Leu and Cys codons in mtd whereas C is usually found at the corresponding wobble positions in other M. thermoautotrophicum genes.

The NH(2)-terminal sequence of the purified protein demonstrated that the translation initiating methionine residue was removed from the mature mtd gene product. The calculated molecular mass for the mtd gene product was 29,644, and this protein was predicted to be hydrophobic and acidic with a pI of 4.2 and the net charges at pH 7 and 9 of -11.64 and -15.6, respectively. The MTD protein did not contain tryptophan residues.

Comparison of the nucleotide and amino acid sequences of mtd and ORFX with the sequences available in the GenBank, EMBL (GenEMBL), Swiss-Prot, or PIR protein data bases revealed no recognizable homologies. By aligning the sequences of the beta-subunits of F-reducing hydrogenase (FRHB) from M. thermoautotrophicum DeltaH (24) and the F-reducing formate dehydrogenase (FDHB) from Methanobacterium formicicum(29) , Alex et al.(24) identified a conserved sequence and proposed this as the site of F interaction. Addition of the MTD sequence to this alignment revealed a similar conservation giving a consensus sequence of A - S - D - EI - K - G - GG - VT - LL - - LLDEGI. The consensus improved further (A - S - DI - IAKAG - - GG - VTGLL - FLLDEGI - - - A - AA; Fig. 2) when the amino acid sequence of the deazaflavindependent DNA-photolyase from Anacystis nidulans(30, 31) was added to the alignment.


Figure 2: Alignment of NH(2)-terminal regions of F-dependent enzymes. The PILEUP program of the GCG package was used to generate the alignment shown. Residue numbers include initiator methionines. Sequences: MTD, F-dependent methylene-H(4)MPT dehydrogenase from M. thermoautotrophicum strain Marburg; FRHB, beta-subunit of F-reducing hydrogenase from M. thermoautotrophicum strain DeltaH(24) ; FDHB, beta-subunit of F-reducing formate dehydrogenase from M. formicicum(29) ; PHR, DNA-photolyase from A. nidulans(30, 31) .



Identification of the mtd Transcript and the Site of Transcription Initiation

The 18-mer oligonucleotide 5`-TATAACCTCATCACCCAG-3` complimentary to positions -570 to -553 (Fig. 1B) was used as the ORFX-specific probe and the 19-mer oligonucleotide 5`-GTAACAGGTCCAGCACAGG-3` complimentary to positions 49-67 was used as the mtd-specific probe to probe the Northern blots. The mtd-specific probe hybridized to a 900-nucleotide transcript (Fig. 3), whereas the ORFX-specific probe showed no detectable hybridization (data not shown). The primer extension experiment using the mtd-specific 19-mer oligonucleotide as the primer generated a strong signal corresponding to a transcript initiated at the G located 8 bp upstream from the mtd translation initiating ATG codon (Fig. 4), and a weaker signal was also observed corresponding to mtd transcription initiation occurring at position -9, 1 bp further upstream. Primer extension experiments using the ORFX-specific 18-mer oligonucleotide as the primer and the same preparation of M. thermoautotrophicum Marburg RNA did not give a signal (data not shown).

Synthesis of F-dependent Methylene-H(4)MPT Dehydrogenase in E. coli and Properties of the Recombinant Enzyme

Cell extracts of E. coli (pPM58) contained a methylene-H(4)MPT dehydrogenase activity that was strictly dependent on coenzyme F ( Table 1and data not shown); such an activity was not present in extracts of E. coli XL1-Blue or E. coli (pBluescript) (Table 1). Western blot analyses (Fig. 5) demonstrated the presence of an anti-dehydrogenase antiserum-reactive protein in E. coli (pPM58) cell extracts with an electrophoretic mobility similar to that of the native F-dependent dehydrogenase or MTD from M. thermoautotrophicum Marburg. The specific activity of MTD in M. thermoautotrophicum Marburg cell extracts was 6-16-fold higher than that in E. coli (pPM58) cell extracts. Anaerobic extracts of anaerobically grown E. coli (pPM58) cells had more than 2-fold higher specific activity of MTD than the extracts of aerobically grown E. coli (pPM58). Extracts of E. coli (pBE103) cells neither possessed the MTD activity (Table 1), nor showed any immunoreactive band in Western blots probed with anti-MTD antiserum (data not shown). The anaerobic E. coli cell extracts had no F-independent (hydrogenase-type) methylene-H(4)MPT dehydrogenase activity.




Figure 5: Western blot of SDS-PAGE separated proteins in E. coli and M. thermoautotrophicum Marburg cell extracts. Amounts of extract proteins used: E. coli (pBluescript) cell extract, 80 µg; E. coli (pPM58) cell extract, 80 µg; F-dependent methylene-H(4)MPT dehydrogenase (MTD) purified from M. thermoautotrophicum Marburg, 0.06 µg; M. thermoautotrophicum Marburg cell extract, 4.2 µg. Anti-MTD rabbit antiserum was used as the primary antibody and alkaline phosphatase-conjugated anti-rabbit goat IgG as the secondary antibody.



The MTD activity in E. coli (pPM58) cell extracts was maximum at pH 4.7 and at 45-55 °C, whereas the native enzyme purified from M. thermoautotrophicum Marburg shows maximum activity at pH 4 and at 55-65 °C(4) . The recombinant enzyme was stable for >70 h at 25 and 40 °C, but at 65 °C it lost 45% of its activity in 1.5 h and 93% in 24 h. The native enzyme is stable at 25 and 40 °C but loses 35% of its activity after 2 h and 96% after 27 h at 65 °C(4) .


DISCUSSION

Using an oligonucleotide probe based on the the NH(2)-terminal amino acid sequence of the purified protein, we have cloned and sequenced the gene (mtd) that encodes the F-dependent methylene-H(4)MPT dehydrogenase (MTD) of M. thermoautotrophicum Marburg. The recombinant MTD synthesized in E. coli (pPM58) is oxygen-stable, catalytically active, and dependent on coenzyme F as the electron carrier. It reacts with antibodies raised against the oxygen-stable native MTD purified from M. thermoautotrophicum Marburg(2) , and its pH and temperature optima for activity and heat resistance are only slightly different from those of the native enzyme. The recombinant enzyme must therefore fold correctly in a mesophilic bacterial cell generating an active enzyme. Our data also suggest that MTD does not require a methanogen-specific prosthetic group for activity. Therefore, mutational studies on MTD could be carried out using the recombinant enzyme.

Although the specific activity of the recombinant enzyme was higher in anaerobically grown E. coli (pPM58) than in aerobically grown cells (Table 1), this observation does not indicate an oxygen-sensitive nature for the recombinant MTD, since anaerobic cell extracts retained their original MTD specific activity after exposure to air (data not shown).

During purification from M. thermoautotrophicum, MTD is found to be tightly associated with methyl-coenzyme M methylreductase (32) (^2)and the predicted hydrophobic nature of MTD is consistent with this observation.

The predicted molecular mass of dehydrogenase was about 2.4 kDa lower than that estimated for the purified native protein from M. thermoautotrophicum Marburg by denaturing gel electrophoresis at pH 9(2) . This difference could be attributed to an overestimation of molecular mass in SDS-PAGE, since the deduced net charge per subunit of dehydrogenase at pH 9 is -15.6.

The determined size of the mtd transcript (900 nucleotide; Fig. 3) is consistent with a monocistronic mRNA containing only the 825-bp mtd gene and its immediately flanking regions. The primer extension experiments demonstrated that the mtd transcript initiated primarily at the G nucleotide located 8 bp upstream of the translation initiating ATG codon and consistent with these results the sequence 5`-TTAATAA-3` that is located 22 bp further upstream (Fig. 1B) corresponds both in the location and sequence to that expected for the TATA-box component of a methanogen promoter(33) . The mtd coding sequence is followed by several poly(dT) sequences, and such sequences have been proposed as transcription terminators for many methanogen genes(22, 23, 33, 34, 35, 36) .

The proposed transcription start site for mtd (G at -8) is the 3rd bp of the sequence from position -10 to -3 (indicated in Fig. 1B by asterisks) that is complementary to the sequence at the 3`-end of 16 S rRNA(20) . Obviously, only the transcribed sequence, -8 to -3, could function as a ribosome binding site. An 8-nucleotide-long upstream region in a mRNA, as observed for mtd, is unusually short. A second archaeal mRNA with an extremely short upstream sequence is the bacterio-opsin mRNA of Halobacterium halobium, where the AUG codon is preceded by only two nucleotides(37) . The implications of such short upstream sequences are unclear.

The amino acid sequence determined for MTD has no detectable conservation with the hydrogenase-type enzyme(6) . This observation is consistent with these two enzymes catalyzing mechanistically different reactions(2, 6, 38) .

Alex et al.(24) compared the amino acid sequence of the beta-subunit of FRH from M. thermoautotrophicum DeltaH (24) with that of FDH from M. formicicum(29) and identified a conserved sequence. They speculated that as both enzymes reduce F via bound FADH(2) moieties, the conserved regions are probably involved in this process(24) . MTD does not contain flavin (4) but does interact with F(2, 4) . When we aligned MTD with FRHB and FDHB, a conserved sequence, almost the same as that reported by Alex et al.(24) , was detected. When the sequence of DNA photolyase (30) from A. nidulans (an enzyme with deazaflavin and FADH(2) as prosthetic groups; (31) ) was added to the alignment (Fig. 2), this conservation was again observed, despite the large evolutionary distance between the bacterium A. nidulans and the methanogenic archaea(39) . Therefore, it is plausible that the NH(2)-terminal regions of all these proteins play a role in interaction with F.


FOOTNOTES

*
Research at The University of Iowa was supported by the United States Department of Agriculture through Research Grant 4132008 to the Biotechnology Byproducts Consortium (to L. D.) and research grants from the University of Iowa Graduate School (to B. M. and E. P.) for the use of University Facilities. The research at Ohio State University was supported by Department of Energy Grant DE-FG02-87ER13731 (to J. N. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) report in this paper has been submitted to the Genome Sequence Data Base with accession number(s) L37108[GenBank].

§
Current address: Dept. of Microbiology, University of Illinois, Urbana, IL 61801.

To whom correspondence should be addressed. Tel.: 319-335-7780 or 319-335-4909; Fax: 319-335-4901.

(^1)
The abbreviations used are: MF, methanofuran; F, coenzyme F (a 7,8-didemethyl-8-hydroxy-5-deaza-riboflavin derivative); FDH, F-reducing formate dehydrogenase; FRH, F-reducing hydrogenase; H(2)F, reduced coenzyme F; H(4)MPT, 5,6,7,8-tetrahydromethanopterin; HC=H(4)MPT, N^5,N-methenyl-H(4)MPT; H(2)C=H(4)MPT, N^5,N-methylene-H(4)MPT; HS-CoM, coenzyme M; MES, 2-(N-morpholino)ethanesulfonic acid; MTD, F-dependent methylenetetrahydromethanopterin dehydrogenase; MTH, F-independent (hydrogenase-type) methylenetetrahydromethanopterin dehydrogenase; kb, kilobase pair(s); bp, base pair(s); ORF, open reading frame.

(^2)
B. Mukhopadhyay, unpublished observation.


ACKNOWLEDGEMENTS

We thank Ken Wright for amino acid analysis; Negash Belay, Carla Kuhner, and Steven C. Clegg for critical comments; and Laura Marchiando, Scott Griffin, and Jörk Nölling for technical assistance. B. M. and E. P. thank Susan Pedigo, Gururajan Rajagopal, Basavapatna Rajagopal, Gerald Gerlach, Young-Min Bae, Nancy Ness-Nichols, Wade Nichols, Dana Swenson, Diana Cruden, Nancy Lynch, and Marian Rawlings for providing technical advice and training. B. M. thanks Steven C. Clegg for supervision and Ralph S. Wolfe for support.


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