(Received for publication, September 27, 1994)
From the
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
-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.
Methanogens are strictly anaerobic archaea and they reduce
CO 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-H
MPT
CH
-S-CoM
CH
, where methanofuran (MF),
tetrahydromethanopterin (H
MPT), and coenzyme M (HS-CoM) are
C
-carrying cofactors(1) .
The interconversion of
methylene-HMPT (H
C=H
MPT) and
methenyl-H
MPT (HC
=H
MPT)
is catalyzed by H
C=H
MPT dehydrogenase.
Cell extracts of Methanobacterium thermoautotrophicum strain
Marburg (M. thermoautotrophicum Marburg) exhibit two types of
H
C=H
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
-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.
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-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 NH
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
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).
The NH-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
-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.
Figure 2:
Alignment of NH-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) .
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
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) .
Using an oligonucleotide probe based on the the
NH-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.
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) ()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 -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
.
The nucleotide sequence(s) report in this paper has been submitted to the Genome Sequence Data Base with accession number(s) L37108[GenBank].