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PubMed1992

Methanococcus voltae harbors four gene clusters potentially encoding two [NiFe] and two [NiFeSe] hydrogenases, each of the cofactor F420-reducing or F420-non-reducing types.

Four gene clusters were identified in Methanococcus voltae which probably all encode hydrogenases of the [NiFe] type. One of these contains four genes, including those for the three subunits of the known [NiFeSe] hydrogenase capable of reducing the natural deazaflavin cofactor F420. In a second homologous cluster, the gene encoding the subunit corresponding to that which contains selenium in the known enzyme has a cysteine codon in the relevant position. In addition, two more gene clusters were detected which are very similar both in gene order and sequence to one which encodes a hydrogenase that reduces viologens in Methanobacterium thermoautotrophicum, but whose natural electron acceptor is as yet unknown. Again, in one of these clusters, one of the structural genes, which codes for a hydrogenase subunit containing the putative Ni-binding site, contains a selenocysteine codon. The homologous gene in the other clusters again shows a cysteine codon in the corresponding location. The four gene clusters are closely linked. Those encoding the two selenium-free enzymes are arranged in opposite polarities with a relatively short intergenic region. This arrangement is discussed in terms of a possible joint transcriptional regulation.

Amino Acid Sequence↗
PubMed1996

Purification, properties and primary structure of H2-forming N5 ,N10 -methylenetetrahydromethanopterin dehydrogenase from Methanococcus thermolithotrophicus.

H2-Forming N5,N10 -methylenetetrahydromethanopterin dehydrogenase (Hmd) is a novel type of hydrogenase found in methanogenic Achaea that contains neither nickel nor iron-sulfur clusters. The enzyme has previously been characterized from Methanobacterium thermoautotrophicum and from Methanopyrus kandleri. We report here on the purification and properties of the enzyme from Methanococcus thermolithotrophicus. The hmd gene was cloned and sequenced. The results indicate that the enzyme from Mc. thermolithotrophicus is functionally and structurally closely related to the H2-forming methylene tetrahydromethanopterin dehydrogenase from Mb. thermoautotrophicum and Mp. kandleri. From amino acid sequence comparisons of the three enzymes, a phylogenetic tree was deduced that shows branching orders similar to those derived from sequence comparisons of the 16S rRNA of the orders Methanococcales, Methanobacteriales, and Methanopyrales.

Amino Acid Sequence↗
PubMed1995

Use of the Escherichia coli uidA gene as a reporter in Methanococcus voltae for the analysis of the regulatory function of the intergenic region between the operons encoding selenium-free hydrogenases.

The Escherichia coli beta-glucuronidase gene uidA was linked to a region of the Methanococcus voltae genome containing the putative promoter of a gene for a DNA-binding protein and introduced into the M. voltae chromosome. It was found that the enzyme was expressed in the cells in easily measurable amounts. The reporter gene was then placed under the control of the intergenic region found between two divergently transcribed gene groups encoding selenium-free hydrogenases, which are measurably transcribed only after selenium depletion. This region is supposed not only to contain the divergent promoters governing the transcription of the hydrogenase genes but also cis regulatory elements necessary for the negative transcriptional regulation in which selenium is involved. It was shown that the intergenic region functioned as a promoter region for the reporter gene in either orientation. The additional finding that beta-glucuronidase expression was dependent on selenium depletion localizes the cis regulatory elements to the intergenic region between the two hydrogenase operons.

Base Sequence↗
PubMed1998

Sequence of archaeal Methanococcus jannaschii alpha-amylase contains features of families 13 and 57 of glycosyl hydrolases: a trace of their common ancestor?

Two sequentially different, seemingly unrelated alpha-amylase families exist, known as family-13 and family-57 glycosyl hydrolases. Despite the common enzyme activity, it has as yet been impossible to detect any sequence similarity between the two families. The detailed analysis of the recently determined sequence of the alpha-amylase from methanogenic archaeon Methanococcus jannaschii using the sensitive Hydrophobic Cluster Analysis method revealed that this alpha-amylase contains features of both families of alpha-amylases. Thus the M. jannaschii alpha-amylase is similar to the Pyrococcus furiosus alpha-amylase from family 57 while it obviously contains most of the sequence fingerprints characteristic for alpha-amylase family 13. Importantly, a glutamic acid residue equivalent with the family-13 catalytic glutamate positioned in the beta 5-strand segment was identified in members of family 57. The results presented in this report indicate that the two families, 13 and 57, are either the products of a very distant common ancestor or have evolved from each other, although at present they can represent two different alpha-amylase families with evolved different catalytic mechanisms, catalytic machinery and folds.

Amino Acid Sequence↗
PubMed2001

Biochemical confirmation and characterization of the family-57-like alpha-amylase of Methanococcus jannaschii.

The gene encoding a family-57-like alpha-amylase in the hyperthermophilic archaeon Methanococcus jannaschii, has been cloned into Escherichia coli. Extremely thermoactive alpha-amylase was confirmed in partially purified enzyme solution of the recombinant culture. This enzyme activity had a temperature optimum of 120 degrees C and a pH optimum 5.0-8.0. The amylase activity is extremely stable against denaturants. Hydrolysis of large sugar polymers with alpha-1-6 and alpha-1-4 linkages yields products including glucose polymers of 1-7 units. Highest activity is exhibited on amylose. The catalyst exhibited a half-life of 50 h at 100 degrees C, among the highest reported thermostabilities of natural amylases.

Amino Acid Sequence↗
PubMed2001

Novel, thermostable family-13-like glycoside hydrolase from Methanococcus jannaschii.

A novel glycoside hydrolase from the hyperthermophilic archaeon Methanococcus jannaschii has been cloned into Escherichia coli. Extremely thermoactive and thermostable amylolytic activity was confirmed in partially purified enzyme solution. This enzyme exhibited a temperature optimum of 100 degrees C and a pH optimum pH 5.0-8.0. Hydrolysis of large 1,6-alpha- and 1,4-alpha-linked polysaccharides yielded glucose polymers of 1-7 units. Incubation with amylose displayed the highest activity. The catalyst was activated and stabilized by Ca2+ and exhibited extreme thermostability at 100 degrees C with a half-life of 78 h.

Amino Acid Sequence↗
PubMed2002

Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis.

During growth of the methanogenic archaeon Methanococcus maripaludis on alanine as the sole nitrogen source under H(2)/CO(2), alanine was incorporated into amino acids derived from pyruvate including leucine, isoleucine, and valine. Thus, growth with alanine was an efficient means of labeling intracellular pools of pyruvate in this lithotroph. Cells were grown with 18% [U-(13)C]alanine, and the distribution of the isotope in the branched-chain amino acids was determined by (13)C-NMR. Carbons derived from pyruvate contained 14.5% (13)C, indicating that most of the cellular pyruvate was obtained from alanine. In contrast, carbons derived from acetyl-CoA contained only 3-5% (13)C, indicating that only small amounts of acetyl-CoA were formed from pyruvate. Thus, autotrophic acetyl-CoA biosynthesis continued even in the presence of an organic carbon source. Moreover, the labeling of acetyl-CoA was lower than would be predicted if pyruvate was a C-1 donor for acetyl-CoA biosynthesis. Carbon derived from the C-1 of acetyl-CoA contained less (13)C than carbon derived from the C-2 of acetyl-CoA, and this difference was attributed to the acetyl-CoA:CO(2) exchange activity of acetyl-CoA synthase. No enrichment was detected for the C-1 of valine, which was derived from the C-1 of pyruvate. This result was attributed to the pyruvate:CO(2) exchange activity of pyruvate oxidoreductase and may have important implications for isotope tracer studies utilizing pyruvate. Lastly, these results demonstrate that the breakdown of pyruvate by methanococci is very limited even under conditions where it is the sole nitrogen and major carbon source.

Alanine↗
PubMed2003

The anabolic pyruvate oxidoreductase from Methanococcus maripaludis.

In autotrophic methanogens, pyruvate oxidoreductase (POR) plays a key role in the assimilation of CO(2) and the biosynthesis of organic carbon. This enzyme has been purified to homogeneity, and the genes from Methanococcus maripaludis were sequenced. The purified POR contained five polypeptides with molecular masses of 47, 33, 25, 21.5 and 13 kDa. The N-terminal sequences of four of the polypeptides had high similarity to the subunits commonly associated with this enzyme from other archaea. However, the 21.5-kDa polypeptide had not been previously observed in PORs. Nucleotide sequencing of the gene cluster encoding the POR revealed six open reading frames ( porABCDEF). The genes porABCD corresponded to the subunits previously identified in PORs. On the basis of the N-terminal amino acid sequence, porE encoded the 21.5-kDa polypeptide and contained a high cysteinyl residue content and a motif indicative of a [Fe-S] cluster. porF also had a high sequence similarity to porE, a high cysteinyl residue content, and two [Fe-S] cluster motifs. Homologs to porE were also present in the genomic sequences of the autotrophic methanogens Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus. Based upon these results, it is proposed that PorE and PorF are components of a specialized system required to transfer low-potential electrons for pyruvate biosynthesis. Some biochemical properties of the purified methanococcal POR were also determined. This unstable enzyme was very sensitive to O(2 )and demonstrated high activity with pyruvate, oxaloacetate, and alpha-ketobutyrate. Methyl viologen, rubredoxin, FMN, and FAD were readily reduced. Activity was also observed with spinach and clostridial ferredoxins and cytochrome c. Coenzyme F(420) was not an electron acceptor for the purified enzyme.

Amino Acid Sequence↗
PubMed2000

Influence of the fusion of two subunits of the F420-non-reducing hydrogenase of Methanococcus voltae on its biochemical properties.

In Methanococcus voltae, one of the two [NiFeSe] hydrogenases is unusual in that the large subunit is split into two subunits, each contributing two ligands to the [NiFe] center that catalyzes the heterolytic cleavage of the dihydrogen molecule. We have engineered a fusion of these two subunits. The resulting new enzyme showed no significant difference in hydrogen uptake activity or in the Ni-C or Ni-L EPR spectra compared to the the wild-type enzyme, but exhibited a tenfold increase in both the Km for hydrogen and the Ki for the competitive inhibitor carbon monoxide.

Binding Sites↗
PubMed2000

A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii.

The pyruvate carboxylase (PYC) of the hyperthermophilic, strictly hydrogenotrophic, autotrophic and marine methanarchaeon Methanococcus jannaschii was purified to homogeneity. Optimal activity was at pH 8.5, > or = 80 degrees C, and a KCl concentration of 0.175 M. This enzyme is the most thermophilic PYC so far studied. Unlike the Methanobacterium thermoautotrophicum enzyme, Mc. jannaschii PYC was expressed in cells grown without an external source of biotin and in the purified form was stable during storage at 4, -20 and -80 degrees C. However, it was rapidly inactivated at 80 degrees C. The enzyme was insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and was strongly inhibited by ATP and ADP (apparent Km, for ATP, 0.374 +/- 0.039 mM; apparent Ki for ATP, 5.34 +/- 2.14 mM; Ki for ADP, 0.89 +/- 0.18 mM). It was also strongly inhibited when the Mg2+ concentration in the assay exceeded that of ATP. Thus, this stable PYC could serve as a model for mechanistic studies on archaeal PYCs. It was apparently an alpha4beta4-type PYC composed of a non-biotinylated 55.5-kDa subunit (PYCA) and a 64.2-kDa biotinylated subunit (PYCB). The determined NH2-terminal sequences for these subunits provided additional support for our earlier proposal to rename the ORFs MJ1229 and MJ1231 in the NCBI Mc. jannaschii genome sequence database as PYCA and PYCB, respectively; even very recently, these have been misidentified as a subunit of acetyl-CoA carbxoylase (AccC) and the alpha-subunit of ion-pumping oxaloacetate decarboxylase (OADalpha), respectively.

Adenosine Diphosphate↗
PubMed1998

The vhuU gene encoding a small subunit of a selenium-containing [NiFe]-hydrogenase in Methanococcus voltae appears to be essential for the cell.

We developed a general method for the site-specific deletion of gene sequences to obtain new selectable markers in the archaeon Methanococcus voltae. Using a deletion in the hisA gene, a vector was integrated into the chromosome by homologous recombination, thereby reconstituting histidine prototrophy. The vector contained the beta-glucuronidase gene uidA of Escherichia coli as a reporter under the control of an M. voltae promoter that normally drives the expression of a selenium-free [NiFe]-hydrogenase after selenium deprivation. This construct has allowed us to check whether the selenium supply was sufficiently low to induce the transcription of the genes encoding the selenium-free hydrogenases. We tried to introduce a chromosomal deletion of the vhuU gene of the archaeon M. voltae by gene replacement and by keeping the cells under selenium deprivation. The gene vhuU encodes the very small, selenocysteine-containing subunit that is part of the primary reaction center of the Vhu hydrogenase. All transformants bearing the deletion also contained the vhuU wild-type gene. Therefore, the vhuU gene appears to be essential for the cell even under conditions that lead to the induction of the selenium-free homologue Vhc of the Vhu hydrogenase.

Aldose-Ketose Isomerases↗
PubMed2001

Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing.

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.

Amino Acid Sequence↗
PubMed2001

Novel glucoamylase-type enzymes from Thermoactinomyces vulgaris and Methanococcus jannaschii whose genes are found in the flanking region of the alpha-amylase genes.

A region downstream of the gene for pullulan-hydrolyzing alpha-amylase, TVA II, of Thermoactinomyces vulgaris R-47 was sequenced, and an open reading frame encoding an enzyme homologous to glucoamylase was found. The nucleotide sequence of this enzyme, designated TGA, consists of 1,953 base pairs corresponding to a protein of 651 amino acid residues. The TGA gene was subcloned and expressed in Escherichia coli. Enzymatic analyses showed that, like other glucoamylases, TGA produced beta-D-glucose from its substrate. However, TGA hydrolyzed maltooligosaccharides such as maltotetraose and maltose more efficiently than starch, while fungal glucoamylases preferred starch to maltooligosaccharides. The primary structure of TGA resembled a putative glucoamylase from the hyperthermophilic archaeon Methanococcus jannaschii (MGA), while homologies between TGA and the fungal glucoamylases were low. The enzymatic properties of recombinant MGA produced in E. coli cells were similar to those of TGA. These findings indicate that TGA and MGA are novel glucoamy-lase-type enzymes with oligosaccaharide-metabolizing activity.

Amino Acid Sequence↗
PubMed1998

Flagellin genes of Methanococcus vannielii: amplification by the polymerase chain reaction, demonstration of signal peptides and identification of major components of the flagellar filament.

The highly conserved nature of the 5'-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (Mr = 30,800 and 28,600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins.

Amino Acid Sequence↗
PubMed2001

Insertional inactivation of the flaH gene in the archaeon Methanococcus voltae results in non-flagellated cells.

The marine methanogen Methanococcus voltae possesses two transcriptional units that encode a total of four flagellins. Immediately downstream of the flagellin genes are a number of ORFs, some of which are cotranscribed with the flagellin genes. These putative genes have been named flaCDEFGHIJ, although no biochemical data has implicated them in flagellar morphogenesis. None of the flaC-J genes has homology to any bacterial gene, with the exception of flaI, which shows homology to pilT, a gene that encodes a nucleotide binding protein of the type IV pilus family. In this study, insertional mutations in flaH of M. voltae were identified. The mutants were non-motile and non-flagellated as determined by electron microscopy. Southern hybridization experiments confirmed the insertion of a mutagenic vector into flaH and indicated that two, tandem, copies of the vector were present. It is believed that insertion of the vector into flaH should disrupt the transcription of flaIJ due to polar effects. The flaH mutant displayed the same pattern of multiple mRNA transcripts, all originating upstream of flaB1, as the wild-type cells. Northern hybridization experiments failed to detect a flaHIJ transcript in either wild-type or mutant cells. Immunoblotting experiments indicated, however, that the mutants produced similar amounts of flagellin, FlaD and FlaE to wild-type cells. Flagellin localization experiments suggest that the flaH mutant is deficient in flagellin secretion and/or assembly. The mutant also displayed similar preflagellin peptidase activity to the wild-type cells, indicating that none of the genes flaHIJ is likely to be the gene that encodes this enzyme, which is required for cleaving the leader peptide from the preflagellins prior to their incorporation into the flagellar filament. This is the first data indicating that the flaHIJ gene cluster is essential for flagellation in methanogens.

Archaeal Proteins↗
PubMed2001

Coordinate positive regulation of genes encoding [NiFe] hydrogenases in Methanococcus voltae.

Two transcription units encoding selenium-free [NiFe] hydrogenases in Methanococcus voltae are transcribed only upon selenium deprivation. Their products replace or complement selenocysteine-containing isoenzymes. The transcription units are linked by a 453-bp intergenic region, and are subject to both positive and negative transcriptional regulation. The mechanism of positive regulation was studied in detail. Mutations in identical 11-bp putative activator recognition sites close to each promoter showed that each site is involved in the activation of both promoters. Sequence-specific DNA-affinity chromatography yielded a 55-kDa protein which specifically recognized the 11-bp sequence. We consider this protein to be a transcriptional activator for both transcription units.

Base Sequence↗
PubMed2003

Isolation of a complete A1AO ATP synthase comprising nine subunits from the hyperthermophile Methanococcus jannaschii.

Archaeal A(1)A(O) ATP synthase/ATPase operons are highly conserved among species and comprise at least nine genes encoding structural proteins. However, all A(1)A(O) ATPase preparations reported to date contained only three to six subunits and, therefore, the study of this unique class of secondary energy converters is still in its infancy. To improve the quality of A(1)A(O) ATPase preparations, we chose the hyperthermophilic, methanogenic archaeon Methanococcus jannaschii as a model organism. Individual subunits of the A(1)A(O) ATPase from M. jannaschii were produced in E. coli, purified, and antibodies were raised. The antibodies enabled the development of a protocol ensuring purification of the entire nine-subunit A(1)A(O) ATPase. The ATPase was solubilized from membranes of M. jannaschii by Triton X-100 and purified to apparent homogeneity by sucrose density gradient centrifugation, ion exchange chromatography, and gel filtration. Electron micrographs revealed the A(1) and A(O) domains and the central stalk, but also additional masses which could represent a second stalk. Inhibitor studies were used to demonstrate that the A(1) and A(O) domains are functionally coupled. This is the first description of an A(1)A(O) ATPase preparation in which the two domains (A(1) and A(O)) are fully conserved and functionally coupled.

Adenosine Triphosphatases↗
PubMed2003

Effect of temperature and pressure on the proteolytic specificity of the recombinant 20S proteasome from Methanococcus jannaschii.

The hydrolytic specificity of the recombinant 20S proteasome from the deep-sea thermophile Methanococcus jannaschii was evaluated toward oxidized insulin B-chain across a range of temperatures (35 degrees, 55 degrees, 75 degrees, and 90 degrees C) and hydrostatic pressures (1, 250, 500, and 1,000 atm). Of the four temperatures considered, the same maximum overall hydrolysis rate was observed at both 55 degrees and 75 degrees C, which are much lower than the T(opt) of 116 degrees C previously observed for a small amide substrate (Michels and Clark 1997). At 35 degrees C the rates of cleavage were highest at the carboxyl side of glutamine and leucine, whereas at the three higher temperatures, the most rapid cleavages occurred after leucine and glutamic acid residues. The distribution of proteolytic fragments and the cleavage sequence also varied between the lowest and higher temperatures. Application of hydrostatic pressure did not increase proteasome activity, as observed previously for the amide substrate (Michels and Clark 1997), but instead significantly reduced the overall conversion of the polypeptide substrate. Overall cleavage patterns observed for the recombinant M. jannaschii proteasome were similar to those reported previously for Thermoplasma acidophilum (Akopian et al. 1997) and human proteasomes (Dick et al. 1991), indicating that proteasome specificity has been conserved despite significant environmental diversity.

Amino Acid Sequence↗