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PubMed2001

Detailed analysis of RNA-protein interactions within the ribosomal protein S8-rRNA complex from the archaeon Methanococcus jannaschii.

The crystal structure of ribosomal protein S8 bound to its target 16 S rRNA from a hyperthermophilic archaeon Methanococcus jannaschii has been determined at 2.6 A resolution. The protein interacts with the minor groove of helix H21 at two sites located one helical turn apart, with S8 forming a bridge over the RNA major groove. The specificity of binding is essentially provided by the C-terminal domain of S8 and the highly conserved nucleotide core, characterized by two dinucleotide platforms, facing each other. The first platform (A595-A596), which is the less phylogenetically and structurally constrained, does not directly contact the protein but has an important shaping role in inducing cross-strand stacking interactions. The second platform (U641-A642) is specifically recognized by the protein. The universally conserved A642 plays a pivotal role by ensuring the cohesion of the complex organization of the core through an array of hydrogen bonds, including the G597-C643-U641 base triple. In addition, A642 provides the unique base-specific interaction with the conserved Ser105, while the Thr106 - Thr107 peptide link is stacked on its purine ring. Noteworthy, the specific recognition of this tripeptide (Thr-Ser-Thr/Ser) is parallel to the recognition of an RNA tetraloop by a dinucleotide platform in the P4-P6 ribozyme domain of group I intron. This suggests a general dual role of dinucleotide platforms in recognition of RNA or peptide motifs. One prominent feature is that conserved side-chain amino acids, as well as conserved bases, are essentially involved in maintaining tertiary folds. The specificity of binding is mainly driven by shape complementarity, which is increased by the hydrophobic part of side-chains. The remarkable similarity of this complex with its homologue in the T. thermophilus 30 S subunit indicates a conserved interaction mode between Archaea and Bacteria.

Amino Acid Sequence↗

PubMed1998

Purification, crystallization, and preliminary X-ray crystallographic data analysis of small heat shock protein homolog from Methanococcus jannaschii, a hyperthermophile.

A gene coding for a small heat shock protein homolog from the hyperthermophilic methanogenic Archaeon Methanococcus jannaschii was cloned. This gene was overexpressed in Escherichia coli harboring rare codon tRNAs and its protein purified and crystallized. Crystals displayed the space group R3 with unit cell dimensions a = b = 171.46 A and c = 102.13 A in a hexagonal axis setting. These crystals grew in one week and diffracted to 3.2 A resolution. The presence of eight molecules in the asymmetric unit gives a Vm value of 2.2 A3/Da and a solvent content of 44% by volume. The 24-molecule complex is generated from a subunit by a combination of crystallographic threefold symmetry and three types of noncrystallographic symmetries (a two-, a three-, and a fourfold).

Archaeal Proteins↗

PubMed1998

Crystal structure determination of FtsZ from Methanococcus jannaschii.

FtsZ is the polymer-forming protein of bacterial cell division. It is part of a ring in the middle of the dividing cell that is required for constriction of cell membrane and cell envelope to yield two daughter cells. FtsZ is a GTPase and is the only bacterial protein showing significant sequence homology to the eukaryotic tubulins. FtsZ can polymerize into tubes, sheets, and rings in vitro and is ubiquitous in eubacteria and archaea. Full-length FtsZ1 from Methanococcus jannaschii has been over expressed in Escherichia coli, employing the hyperthermophilic properties of the protein. Crystals grown from PEG400 and ethanol belong to spacegroup I213 with a = b = c = 159.1 A. Isomorphous replacement using one Hg derivative yielded a interpretable electron density map at 4 A resolution. The structure for residues 23-356 and one GDP has been refined to an Rfree of 0.28 (Rf = 0.20) at 2.8 A resolution. FtsZ consists of two domains with a connecting core helix. The N-terminal domain and the core helix contain all residues involved in nucleotide binding and resemble the fold of dinucleotide-binding proteins. The structures of tubulin and FtsZ show striking similarity; together with the functional similarities, this provides a strong indication that FtsZ is a true homolog of tubulin.

Bacterial Proteins↗

PubMed1999

Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.

We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside. The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein had a dimeric structure composed of identical subunits, and the M(r) of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit M(r) was 36, 000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K(m)) for (RS)-mevalonate was 68.5 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.

Amino Acid Sequence↗

PubMed1994

Selenium is involved in the negative regulation of the expression of selenium-free [NiFe] hydrogenases in Methanococcus voltae.

Competitive polymerase chain reactions (PCR) were used to analyze quantitatively the transcription patterns of the four different gene groups encoding [NiFe] hydrogenases in Methanococcus voltae. In cells growing in the presence of selenium, transcripts of only two of the hydrogenase transcription units could be detected in quantities above background levels. The missing transcripts encode the selenium-free F420-non-reducing and F420-reducing hydrogenases. In cells grown without selenium these transcripts are detectable, indicating the involvement of selenium in their transcriptional regulation.

Base Sequence↗

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↗

PubMed2003

Glucose-6-phosphate isomerase from the hyperthermophilic archaeon Methanococcus jannaschii: characterization of the first archaeal member of the phosphoglucose isomerase superfamily.

ORF MJ1605, previously annotated as pgi and coding for the putative glucose-6-phosphate isomerase (phosphoglucose isomerase, PGI) of the hyperthermophilic archaeon Methanococcus jannaschii, was cloned and functionally expressed in Escherichia coli. The purified 80-kDa protein consisted of a single subunit of 45 kDa, indicating a homodimeric (alpha(2)) structure. The K(m) values for fructose 6-phosphate and glucose 6-phosphate were 0.04 mM and 1 mM, the corresponding V(max) values were 20 U/mg and 9 U/mg, respectively (at 50 degrees C). The enzyme had a temperature optimum at 89 degrees C and showed significant thermostability up to 95 degrees C. The enzyme was inhibited by 6-phosphogluconate and erythrose-4-phosphate. RT-PCR experiments demonstrated in vivo expression of ORF MJ1618 during lithoautotrophic growth of M. jannaschii on H(2)/CO(2). Phylogenetic analyses indicated that M. jannaschii PGI was obtained from bacteria, presumably from the hyperthermophile Thermotoga maritima.

Cloning, Molecular↗

PubMed2003

The importance of porE and porF in the anabolic pyruvate oxidoreductase of Methanococcus maripaludis.

The operon of the anabolic pyruvate oxidoreductase (POR) of Methanococcus maripaludis encodes two genes ( porEF) whose functions are unknown. Because these genes possess sequence similarity to polyferredoxins, they may be electron carriers to the POR. To elucidate whether the methanococcal POR requires PorEF for activity, a deletion mutant, strain JJ150, lacking porEF was constructed. Compared to the wild-type strain JJ1, the mutant grew more slowly in minimal medium and minimal plus acetate medium, and pyruvate-dependent methanogenesis was inhibited. In contrast, the methyl-viologen-dependent pyruvate-oxidation activity of POR, carbon monoxide dehydrogenase, and hydrogenase activities of the mutant were similar to those of the wild-type. Upon genetic complementation of the mutant with porEF in the methanococcal shuttle vector pMEV2+ porEF, growth in minimal medium and pyruvate-dependent methanogenesis were restored to wild-type levels. Complementation with porE alone restored methanogenesis from pyruvate but not growth in minimal medium. Complementation with porF alone partially restored growth but not methanogenesis from pyruvate. Although the specific roles of porE and porF have not been determined, these results suggest that PorEF play important roles in the anabolic POR in vivo even though they are not required for the dye-dependent activity.

Aldehyde Oxidoreductases↗

PubMed2004

A pseudo-SECIS element in Methanococcus voltae documents evolution of a selenoprotein into a sulphur-containing homologue.

Methanococcus maripaludis possesses two sets of F(420)-non-reducing hydrogenases which are differentially expressed in response to the selenium content of the medium. One of the subunits of the selenium-containing hydrogenase, VhuD, contains two selenocysteine residues, whereas the homologue of M. voltae possesses cysteine residues in the equivalent positions. Analysis of the 3' non-translated region of the M. voltae vhuD mRNA revealed the existence of a structure resembling the consensus of archaeal SECIS elements but with deviations rendering it non-functional in determining selenocysteine insertion. The presence of a pseudo-SECIS element in the 3' non-translated region of the vhuD mRNA from M. voltae suggests that VhuD from this organism has developed from a selenocysteine-containing ancestor. The 3' non-translated region from the VhcD homologues neither contained a SECIS nor a pseudo SECIS element.

3' Untranslated Regions↗

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↗