Characterization of a hyperthermophilic P-type ATPase from Methanococcus jannaschii expressed in yeast

We report on the biochemical and structural properties of a putative P-type H(+)-ATPase, MJ1226p, from the anaerobic hyperthermophilic Archaea Methanococcus jannaschii. An efficient heterologous expression system was developed in Saccharomyces cerevisiae and a four-step purification protocol, using n-dodecyl beta-d-maltoside, led to a homogeneous detergent-solubilized protein fraction with a yield of over 2 mg of protein per liter of culture. The three-dimensional structure of the purified detergent-solubilized protein obtained at 2.4 nm resolution by electron microscopy showed a dimeric organization in which the size and the shape of each monomer was compatible with the reported structures of P-type ATPases. The purified MJ1226p ATPase was inactive at 40 degrees C and was active at elevated temperature reaching high specific activity, up to 180 micromol of P(i) x min(-1) x mg(-1) at 95 degrees C. Maximum ATPase activity was observed at pH 4.2 and required up to 200 mm monovalent salts. The ATPase activity was stable for several days upon storage at 65 degrees C and was highly resistant to urea and guanidine hydrochloride. The protein formed catalytic phosphoenzyme intermediates from MgATP or P(i), a functional characteristic specific of P-type ATPases. The highly purified, homogeneous, stable, and active MJ1226p ATPase provides a new model for further structure-function studies of P-type ATPases.     

Enzymatic properties of S-adenosylmethionine synthetase from the archaeon Methanococcus jannaschii

S-Adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, MAT) catalyzes a unique enzymatic reaction that leads to formation of the primary biological alkylating agent. MAT from the hyperthermophilic archaeon Methanococcus jannaschii (MjMAT) is a prototype of the newly discovered archaeal class of MAT proteins that are nearly unrecognizable in sequence when compared with the class that encompasses both the eucaryal and bacterial enzymes. In this study the functional properties of purified recombinant MjMAT have been evaluated. The products of the reaction are AdoMet, PP(i), and P(i); >90% of the P(i) originates from the gamma-phosphoryl group of ATP. The circular dichroism spectrum of the dimeric MjMAT indicates that the secondary structure is more helical than the Escherichia coli counterpart (EcMAT), suggesting a different protein topology. The steady state kinetic mechanism is sequential, with random addition of ATP and methionine; AdoMet is the first product released, followed by release of PP(i) and P(i). The substrate specificity differs remarkably from the previously characterized MATs; the nucleotide binding site has a very broad tolerance of alterations in the adenosine moiety. MjMAT has activity at 70 degrees C comparable with that of EcMAT at 37 degrees C, consistent with the higher temperature habitat of M. jannaschii. The activation energy for AdoMet formation is larger than that for the E. coli MAT-catalyzed reaction, in accord with the notion that enzymes from thermophilic organisms are often more rigid than their mesophilic counterparts. The broad substrate tolerance of this enzyme proffers routes to preparation of novel AdoMet analogs.     

ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.     

Kinetic mechanism of fuculose-1-phosphate aldolase from the hyperthermophilic archaeon Methanococcus jannaschii

Fuculose-1-phosphate aldolase (FucA) is a useful biocatalyst with potential applications in chiral synthesis. In this study, the overall kinetic mechanism of FucA from the archaeon Methanococcus jannaschii was studied. The K(m) values of dihydroxyacetone phosphate (DHAP) and dl-glyceraldehyde were 0.09 and 0.74 mM, respectively. Dead-end inhibition by trimethyl phosphonoacetate and dl-threose were competitive and uncompetitive with respect to DHAP and dl-glyceraldehyde. Inhibition patterns obtained using reaction products were noncompetitive vs. DHAP and competitive vs. dl-glyceraldehyde. The equilibrium constant was 8.309×10(-3) M as assessed by varying the [DHAP]/[product] ratio at a fixed dl-glyceraldehyde concentration and by measuring the change in DHAP concentration after equilibrium was reached. This constant is consistent with the K(eq) value obtained from (13)C NMR (15.625×10(-3) M). The resultant inhibition kinetics may suggest the insights of kinetic mechanism of the FucA catalyzed reaction.     

Identification and characterisation of the selenocysteine-specific translation factor SelB from the archaeon Methanococcus jannaschii

Selenocysteine insertion into archaeal selenopolypeptides is directed through an mRNA structure (the SECIS element) situated in the 3' non-translated region like in eukaryotes. To elucidate the mechanism how this element affects decoding of an in-frame UGA with selenocysteine the open reading frames of the genome of Methanococcus jannaschii were searched for the existence of a homolog to the bacterial specialized translation factor SelB. The product of the open reading frame MJ0495 was identified as the archaeal SelB homolog on the basis of the following characteristics: (1) MJ0495 possesses sequence features characteristic of bacterial SelB; (2) purified MJ0495 displays guanine nucleotide binding properties like SelB; and (3) it preferentially binds selenocysteyl-tRNA(Sec). In contrast to bacterial SelB, however, no binding of MJ0495 protein to the SECIS element of the mRNA was found under the experimental conditions employed which correlates with the fact that MJ0495 lacks the C-terminal domain of the bacterial SelB protein known to bind the SECIS element. It is speculated that in Archaea the functions of bacterial SelB are distributed over at least two proteins, one, serving as the specific translation factor, like MJ0495, and another one, binding to the SECIS which interacts with the ribosome and primes it to decode UGA.     

Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii.

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.     

A thioredoxin from the hyperthermophilic archaeon Methanococcus jannaschii has a glutaredoxin-like fold but thioredoxin-like activities

A thioredoxin homologue (Mj0307) from the hyperthermophilic archaeon Methanococcus jannaschii (MjTRX) was cloned, produced in E. coli, and compared to the thioredoxin from E. coli (ETRX). The secondary structure profile of MjTRX obtained by NMR spectroscopy shows that it has four beta-sheets and three alpha-helices arranged in betaalphabetaalphabetabetaalpha, similar to that of glutaredoxin. However, MjTRX supports the growth of T7 bacteriophage in E. coli and is weakly reduced by the thioredoxin reductase from E. coli, indicating that MjTRX is functionally closer to a thioredoxin than a glutaredoxin. MjTRX has higher specific insulin reductase activity than ETRX and retained its full activity over 4 days at 95 degrees C, whereas ETRX lost its activity in 150 min. The standard state redox potential of MjTRX is about -277 mV, which is the lowest value thus far known among redox potentials of the thioredoxin superfamily. This indicates that the lower redox potential is necessary in keeping catalytic disulfide bonds reduced in the cytoplasm and in coping with oxidative stress in an anaerobic hyperthermophile.     

Translation termination factor aRF1 from the archaeon Methanococcus jannaschii is active with eukaryotic ribosomes

A bacterioferritin was recently isolated from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841–6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron-coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate-reducing bacteria.     

Characterization of a P-type ATPase of the archaebacterium Methanococcus voltae

The vanadate-sensitive ATPase of Methanococcus voltae has been purified by a procedure which includes, purification of the cytoplasmic membrane by sucrose gradient centrifugation, solubilization with Triton X-100, and DEAE-Sephadex and Sephacryl S-300 chromatography. While the DEAE-Sephadex step provided a preparation consisting of two polypeptides (74 and 52 kDa), the Sephacryl S-300 step yields a product with a subunit of 74 kDa. Incubation of either membranes or purified ATPase with [gamma-32P]ATP followed by acidic (pH 2.4) lithium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the vanadate-sensitive labeling of a 74-kDa acyl phosphate intermediate. These results indicate that the M. voltae ATPase is of the P-type.     

S-Adenosylmethionine decarboxylase from the archaeon Methanococcus jannaschii: identification of a novel family of pyruvoyl enzymes

Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems. S-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and Escherichia coli. Despite spermidine concentrations in the Methanococcales that are several times higher than in E. coli, no AdoMet decarboxylase gene was recognized in the complete genome sequence of Methanococcus jannaschii. The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the E. coli speD gene (less than 11% identity). The M. jannaschii enzyme has been expressed in E. coli and purified to homogeneity. Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (alphabeta)(2) complex. The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases. The sequence of the M. jannaschii enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria. The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage. Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes.     

The Methanocaldococcus jannaschii protein Mj0968 is not a P-type ATPase

The Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) protein Mj0968 has been reported to represent a soluble P-type ATPase [Ogawa et al., FEBS Lett. 471 (2000) 99-102]. In this study, we report that the heterologously expressed Mj0968-His(10) protein exhibits high rates of phosphatase activity, whereas only very low ATPase activity was measured. Replacement of the aspartate residue in the DSAGT motif (D7A), which becomes phosphorylated during the reaction cycle of P-type ATPases, does not affect the V(max), but only the K(M) of the reaction. Labeling studies with [gamma-(32)P]ATP and [alpha-(32)P]ATP revealed that the previously reported labeling experiments [Ogawa et al., 2000] do not necessarily show phosphorylation of Mj0968, but rather point to ATP binding. Binding studies with trinitrophenyl adenosine nucleotides showed low apparent K(d) values for those molecules. These results provide evidence that the native function of Mj0968 seems to be that of a phosphatase, rather than that of an ATP-hydrolyzing enzyme.     

Helical tubes of FtsZ from Methanococcus jannaschii

Bacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric forms in vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein from M. jannaschii to produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(alpha,beta)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ from M. jannaschii that were shown to consist of 'thick'-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++ or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the 'thick'-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of approximately 430 A for Ca++-induced tubes or 590 - 620 A for GMPCPP. However, their assembly in vitro as well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a role in vivo. Their size and stability make them suitable for use in motility assays.     

Nucleoid structure and partition in Methanococcus jannaschii: an archaeon with multiple copies of the chromosome.

We measured different cellular parameters in the methanogenic archaeon Methanococcus jannaschii. In exponential growth phase, the cells contained multiple chromosomes and displayed a broad variation in size and DNA content. In most cells, the nucleoids were organized into a thread-like network, although less complex structures also were observed. During entry into stationary phase, chromosome replication continued to termination while no new rounds were initiated: the cells ended up with one to five chromosomes per cell with no apparent preference for any given DNA content. Most cells in stationary phase contained more than one genome equivalent. Asymmetric divisions were detected in stationary phase, and the nucleoids were found to be significantly more compact than in exponential phase.     

Crystal structure of LeuD from Methanococcus jannaschii

3-Isopropylmalate/citramalate (IPM) isomerase catalyzes the second step in the leucine biosynthesis pathway. IPM isomerase from Methanococcus jannaschii is a complex protein consisting of a large (MjLeuC) and a small subunit (MjLeuD). It has broad substrate specificity, unlike other bacterial IPM isomerases. In order to understand the reasons for this broad substrate specificity, we determined the crystal structure of MjLeuD at a resolution of 2.0 Å. The asymmetric unit contained 6 molecules of LeuD, including three homodimers. The overall structure had a β/β/α sandwich-fold consisting of 8 α-helices and 7 β-strands. The C-terminal helix, which is important in homodimer formation, showed conformational differences between two homodimer forms of MjLeuD. In addition, we identified a hydrophobic residue (Val28) near the substrate recognition region that may explain the broad substrate specificity of IPM isomerase. Therefore, we suggest that LeuD proteins can be divided into 2 subfamilies, LeuD subfamilies 1 and 2, which show differences in overall structure and in the substrate recognition region.     

Continuous culture of Methanococcus jannaschii, an extremely thermophilic methanogen

Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a deep-sea hydrothermal vent, was grown at 80 degrees C in continuous culture on a mineral salts medium gassed with H(2) and CO(2) at three different flow rates. The maximum specific growth rate was 0.56 h(-1), and the maximum specific methane productivity was 0.32 (mol g(-1) h(-1)). Uncoupling of growth and methane production was evidenced by an increase in teh non-growth-associated rate of methane formation, beta, with increasing gaseous input. The specific hydrogenase activity exhibited growth-assiciated behaviour at low growth rates, but showed no dependence on growth at higher growth rates. The growth dependence of hydrogenase activity is consistent with the pressure dependence of hydrogenase activity measured in previous experiments. In contrast, the specific protease activity was independent of the growth rate over the entire range of dilution rates studied. (c) 1994 John Wiley & Sons, Inc.     

MJ0917 in archaeon Methanococcus jannaschii is a novel NADP phosphatase/NAD kinase

NAD kinase phosphorylates NAD(+) to form NADP(+). Conversely, NADP phosphatase, which has not yet been identified, dephosphorylates NADP(+) to produce NAD(+). Among the NAD kinase homologs, the primary structure of MJ0917 of hyperthermophilic archaeal Methanococcus jannaschii is unique. MJ0917 possesses an NAD kinase homologous region in its C-terminal half and an inositol-1-phosphatase homologous region in its N-terminal half. In this study, MJ0917 was biochemically shown to possess both NAD kinase and phosphatase activities toward NADP(+), NADPH, and fructose 1,6-bisphosphate, but not toward inositol 1-phosphate. With regard to the phosphatase activity, kinetic values indicated that NADP(+) is the preferred substrate and that MJ0917 would function as a novel NADP phosphatase/NAD kinase showing conflicting dual activities, viz. synthesis and degradation of an essential NADP(+). Furthermore, in vitro analysis of MJ0917 showed that, although MJ0917 could supply NADP(+), it prevented excess accumulation of NADP(+); thus, it has the ability to maintain a high NAD(+)/NADP(+) ratio, whereas 5'-AMP would decrease this ratio. The evolutionary process during which MJ0917 arose is also discussed.     

Characterization of an 8-oxoguanine DNA glycosylase from Methanococcus jannaschii.

A thermostable 8-oxoguanine (oxoG) DNA glycosylase from Methanococcus jannaschii has been expressed in Escherichia coli, purified, and characterized. The enzyme, which has been named mjOgg, belongs to the same diverse DNA glycosylase superfamily as the 8-oxoguanine DNA glycosylases from yeast (yOgg1) and human (hOgg1) but is substantially different in sequence. In addition, unlike its eukaryotic counterparts, which have a strong preference for oxoG.C base pairs, mjOgg has little specificity for the base opposite oxoG. mjOgg has both DNA glycosylase and DNA lyase (beta-elimination) activity, and the combined glycosylase/lyase activity occurs at a rate comparable with the glycosylase activity alone. Mutation of Lys-129, analogous to Lys-241 of yOgg1, abolishes glycosylase activity.     

Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii

The genes from the thermophilic archaeabacterium Methanococcus jannaschii that code for the putative catalytic and regulatory chains of aspartate transcarbamoylase were expressed at high levels in Escherichia coli. Only the M. jannaschii PyrB (Mj-PyrB) gene product exhibited catalytic activity. A purification protocol was devised for the Mj-PyrB and M. jannaschii PyrI (Mj-PyrI) gene products. Molecular weight measurements of the Mj-PyrB and Mj-PyrI gene products revealed that the Mj-PyrB gene product is a trimer and the Mj-PyrI gene product is a dimer. Preliminary characterization of the aspartate transcarbamoylase from M. jannaschii cell-free extract revealed that the enzyme has a similar molecular weight to that of the E. coli holoenzyme. Kinetic analysis of the M. jannaschii aspartate transcarbamoylase from the cell-free extract indicates that the enzyme exhibited limited homotropic cooperativity and little if any regulatory properties. The purified Mj-catalytic trimer exhibited hyperbolic kinetics, with an activation energy similar to that observed for the E. coli catalytic trimer. Homology models of the Mj-PyrB and Mj-PyrI gene products were constructed based on the three-dimensional structures of the homologous E. coli proteins. The residues known to be critical for catalysis, regulation, and formation of the quaternary structure from the well characterized E. coli aspartate transcarbamoylase were compared.     

Activation mechanism of HSP16.5 from Methanococcus jannaschii

The small heat shock proteins are the ubiquitous proteins found in a wide range of organisms and function as molecular chaperones by binding to the folding intermediates of their substrates. Although the crystal structure of HSP16.5, a small heat shock protein from Methanococcus jannaschii, revealed that it is a hollow sphere composed of 24 identical subunits, its activation mechanism remains unclear. We found out that HSP16.5 is active only at high temperatures and forms a stable complex with substrate in a stoichiometric manner. We also observed that the conformational change of HSP16.5 is correlated with the increasing hydrophobic site and its activation as a molecular chaperone. However, it is revealed that the conformational change is not accompanied with the change of the secondary structure of a subunit, but correlated with the increasing diameter of HSP16.5. Therefore, it is proposed that the activation mechanism of HSP16.5 involves temperature induced conformational change with size increment of the complex resulting in the exposure of hydrophobic substrate-binding site.     

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.