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(Received for publication, September 27, 1994) From the
Two methylenetetrahydromethanopterin dehydrogenases have been
purified from Methanobacterium thermoautotrophicum strain
Marburg: one (MTD) is coenzyme F Methanogens are strictly anaerobic archaea and they reduce
CO The interconversion of
methylene-H
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).
Figure 1:
Restriction map and nucleotide sequence
of F
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
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
The DNA sequence was
determined for the cloned F
The NH 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
Figure 2:
Alignment of NH
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
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 Using an oligonucleotide probe based on the the
NH 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) ( 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 ( 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 The nucleotide
sequence(s) report in this paper has been submitted to the Genome
Sequence Data Base with accession number(s)
L37108[GenBank].
Volume 270,
Number 6,
Issue of February 10, 1995 pp. 2827-2832
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-dependent
Methylene-5,6,7,8-tetrahydromethanopterin Dehydrogenase Gene from Methanobacterium thermoautotrophicum Strain Marburg and
Functional Expression in Escherichia coli(*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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
-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
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.
to methane using the following pathway(1) :
CO formyl-MF (
) N
-formyl-H
MPT N
,N-methenyl-H
MPT
N
,N-methylene-H
MPT
N
-methyl-HMPT
CH
-S-CoM CH
, where methanofuran (MF),
tetrahydromethanopterin (HMPT), and coenzyme M (HS-CoM) are
C-carrying cofactors(1) .MPT (HC=HMPT) and
methenyl-HMPT (HC=H
MPT)
is catalyzed by HC=HMPT dehydrogenase.
Cell extracts of Methanobacterium thermoautotrophicum strain
Marburg (M. thermoautotrophicum Marburg) exhibit two types of
HC=HMPT 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
-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.
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
+ CO as
described previously (8) except the media contained 10 mM KHPO, 15 mM KHPO, 0.1 mM trisodium
nitrilotriacetate, and 50 µM FeCl
6HO(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
MPT, and
F-dependent N
,N-methylene-H
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
F, and
HC
=H
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
Purified F-terminal Sequence of
F-dependent Methylene-H
MPT
Dehydrogenase-dependent
H
C= HMPT 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
C=HMPT
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) .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 
-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.
Cloning and Sequencing of the F
The
NH-dependent
Methylene-H
MPT Dehydrogenase Gene (mtd)-terminal amino acid sequence determined for the
F-dependent H
C=HMPT
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).
-dependent methylene-H
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 NHterminal 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.
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.
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).
-dependent
methylene-H
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.-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. -subunits of F-reducing hydrogenase (FRHB) from M. thermoautotrophicum
H (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.
-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
MPT dehydrogenase from M. thermoautotrophicum strain Marburg; FRHB, -subunit of
F-reducing hydrogenase from M. thermoautotrophicum strain
H(24) ; FDHB, -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
Cell extracts of E. coli (pPM58) contained a methylene-H-dependent
Methylene-H
MPT Dehydrogenase in E. coli and Properties
of the Recombinant EnzymeMPT 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
MPT dehydrogenase activity.
-dependent methylene-H
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.
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) .
-terminal amino acid sequence of the purified protein, we
have cloned and sequenced the gene (mtd) that encodes the
F-dependent methylene-H
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.
)and the predicted hydrophobic
nature of MTD is consistent with this observation.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) .
-subunit
of FRH from M. thermoautotrophicum H (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
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
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-terminal regions of
all these proteins play a role in interaction with F.
), coenzyme
F
(a 7,8-didemethyl-8-hydroxy-5-deaza-riboflavin
derivative); FDH, F
-reducing formate dehydrogenase; FRH,
F
-reducing hydrogenase; H
F,
reduced coenzyme F
; H
MPT,
5,6,7,8-tetrahydromethanopterin;
HC=H
MPT, N,N-methenyl-H
MPT;
HC=HMPT, N,N-methylene-H
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.
)
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.
MPT Dehydrogenases of Methanobacterium thermoautotrophicum Strain Marburg and Methanosarcina barkeri and Effects of Methanogenic Substrates on the Levels of Three Catabolic Enzymes in Methanosarcina barkeri . Ph.D. dissertation, University of Iowa, Iowa City, IA
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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