A broad specificity nucleoside kinase from Thermoplasma acidophilum.

The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/ß/α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, fructokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had been annotated as a ribokinase-like protein. Using a coupled pyruvate kinase/lactate dehydrogenase assay, the activity of Ta0880 was assessed against a variety of ribokinase/pfkB-like family substrates; activity was not observed for ribose, fructose-1-phosphate, or fructose-6-phosphate. Based on structural similarity with nucleoside kinases (NK) from Methanocaldococcus jannaschii (MjNK, PDB 2C49, and 2C4E) and Burkholderia thailandensis (BtNK, PDB 3B1O), nucleoside kinase activity was investigated. Ta0880 (TaNK) was confirmed to have nucleoside kinase activity with an apparent KM for guanosine of 0.21 µM and catalytic efficiency of 345,000 M(-1) s(-1) . These three NKs have significantly different substrate, phosphate donor, and cation specificities and comparisons of specificity and structure identified residues likely responsible for the nucleoside substrate selectivity. Phylogenetic analysis identified three clusters within the PfkB family and indicates that TaNK is a member of a new sub-family with broad nucleoside specificities. Proteins 2013. © 2012 Wiley Periodicals, Inc.

Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide.

Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.

Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

Mass spectrometric identification of an intramolecular disulfide bond in thermally inactivated triosephosphate isomerase from a thermophilic organism Methanocaldococcus jannaschii.

The triosephosphate isomerase from the hyperthermophilic organism Methanocaldococcus jannaschii (MjTIM) is a tetrameric enzyme, with a monomer molecular mass of 23245 Da. The kinetic parameters, the k(cat) and the K(m) values, of the enzyme, examined at 25 °C and 50 °C, are 4.18 × 10(4) min(-1) and 3.26 × 10(5) min(-1) , and 0.33 and 0.86 mM(-1) min(-1) , respectively. Although the circular dichroism and fluorescence emission spectra of the protein remain unchanged up to 95 °C, suggesting that the secondary and tertiary structures are not lost even at this extreme temperature, surprisingly, incubation of this thermophilic enzyme at elevated temperature (65-85 °C) results in time-dependent inactivation, with almost complete loss of activity after 3 h at 75 °C. High-resolution electrospray ionization mass spectrometry (ESI-MS) reveals the monomeric mass of the heated sample to be 23243 Da. The 2 Da difference between native and heated samples suggests a probable formation of a disulfide bridge between proximal cysteine thiol groups. Liquid chromatography (LC)/ESI-MS/MS analysis of tryptic digests in the heated samples permits identification of a pentapeptide (DCGCK, residues 80-84) in which a disulfide bond formation between Cys81 and Cys83 was established through the collision-induced dissociation (CID) fragmentation of the intact disulfide-bonded molecule, yielding characteristic fragmentation patterns with key neutral losses. Neither residue is directly involved in the catalytic activity. Inspection of the three-dimensional structure suggests that subtle conformation effects transmitted through a network of hydrogen bonds to the active site residue Lys8 may be responsible for the loss of catalytic activity.

Formation of m2G6 in Methanocaldococcus jannaschii tRNA catalyzed by the novel methyltransferase Trm14.

The modified nucleosides N(2)-methylguanosine and N(2)(2)-dimethylguanosine in transfer RNA occur at five positions in the D and anticodon arms, and at positions G6 and G7 in the acceptor stem. Trm1 and Trm11 enzymes are known to be responsible for several of the D/anticodon arm modifications, but methylases catalyzing post-transcriptional m(2)G synthesis in the acceptor stem are uncharacterized. Here, we report that the MJ0438 gene from Methanocaldococcus jannaschii encodes a novel S-adenosylmethionine-dependent methyltransferase, now identified as Trm14, which generates m(2)G at position 6 in tRNA(Cys). The 381 amino acid Trm14 protein possesses a canonical RNA recognition THUMP domain at the amino terminus, followed by a γ-class Rossmann fold amino-methyltransferase catalytic domain featuring the signature NPPY active site motif. Trm14 is associated with cluster of orthologous groups (COG) 0116, and most closely resembles the m(2)G10 tRNA methylase Trm11. Phylogenetic analysis reveals a canonical archaeal/bacterial evolutionary separation with 20-30% sequence identities between the two branches, but it is likely that the detailed functions of COG 0116 enzymes differ between the archaeal and bacterial domains. In the archaeal branch, the protein is found exclusively in thermophiles. More distantly related Trm14 homologs were also identified in eukaryotes known to possess the m(2)G6 tRNA modification.

The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA.

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

Crystal structure of Mj1640/DUF358 protein reveals a putative SPOUT-class RNA methyltransferase.

The proteins in DUF358 family are all bacterial proteins, which are ∼200 amino acids long with unknown function. Bioinformatics analysis suggests that these proteins contain several conserved arginines and aspartates that might adopt SPOUT-class fold. Here we report crystal structure of Methanocaldococcus jannaschii DUF358/Mj1640 in complex with S-adenosyl-L-methionine (SAM) at 1.4 Å resolution. The structure reveals a single domain structure consisting of eight-stranded ß-sheets sandwiched by six α-helices at both sides. Similar to other SPOUT-class members, Mj1640 contains a typical deep trefoil knot at its C-terminus to accommodate the SAM cofactor. However, Mj1640 has limited structural extension at its N-terminus, which is unique to this family member. Mj1640 forms a dimer, which is mediated by two parallel pairs of α-helices oriented almost perpendicular to each other. Although Mj1640 shares close structural similarity with Nep1, the significant differences in N-terminal extension domain and the overall surface charge distribution strongly suggest that Mj1640 might target a different RNA sequence. Detailed structural analysis of the SAM-binding pocket reveals that Asp157 or Glu183 from its own monomer or Ser43 from the associate monomer probably plays the catalytic role for RNA methylation.

Disulfide linkage in the coiled-coil domain of subunit H of A1AO ATP synthase from Methanocaldococcus jannaschii and the NMR structure of the C-terminal segment H(85-104).

The C-terminal residues 98-104 are important for structure stability of subunit H of A(1)A(O) ATP synthases as well as its interaction with subunit A. Here we determined the structure of the segment H(85-104) of H from Methanocaldococcus jannaschii, showing a helix between residues Lys90 to Glu100 and flexible tails at both ends. The helix-helix arrangement in the C-terminus was investigated by exchange of hydrophobic residues to single cysteine in mutants of the entire subunit H (H(I93C), H(L96C) and H(L98C)). Together with the surface charge distribution of H(85-104), these results shine light into the A-H assembly of this enzyme.

Binding of S-methyl-5'-thioadenosine and S-adenosyl-L-methionine to protein MJ0100 triggers an open-to-closed conformational change in its CBS motif pair.

Cystathionine beta-synthase (CBS) domains are small motifs that are present in proteins with completely different functions. Several genetic diseases in humans have been associated with mutations in their sequence, which has made them promising targets for rational drug design. The protein MJ0100 from Methanocaldococcus jannaschii includes a DUF39 domain of so far unknown function and a CBS domain pair (Bateman domain) at its C-terminus. This work presents the crystallographic analysis of four different states of the CBS motif pair of MJ0100 in complex with different numbers of S-adenosyl-L-methionine (SAM) and S-methyl-5'-thioadenosine (MTA) ligands, providing evidence that ligand-induced conformational reorganization of Bateman domain dimers could be an important regulatory mechanism. These observations are in contrast to what is known from most of the other Bateman domain structures but are supported by recent studies on the magnesium transporter MgtE. Our structures represent the first example of a CBS domain protein complexed with SAM and/or MTA and might provide a structural basis for understanding the molecular mechanisms regulated by SAM upon binding to the C-terminal domain of human CBS, whose structure remains unknown.

Backbone resonance assignments of the 48 kDa dimeric putative 18S rRNA-methyltransferase Nep1 from Methanocaldococcus jannaschii.

Nep1 from Methanocaldococcus jannaschii is a 48 kDa dimeric protein belonging to the SPOUT-class of S-adenosylmethionine dependent RNA-methyltransferases and acting as a ribosome assembly factor. Mutations in the human homolog are the cause of Bowen-Conradi syndrome. We report here 1H, 15N and 13C chemical shift assignments for the backbone of the protein in its apo state.

Biochemical characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase (CobY) enzyme of the hyperthermophilic archaeon Methanocaldococcus jannaschii.

The archaeal cobY gene encodes the nonorthologous replacement of the bacterial NTP:AdoCbi kinase (EC 2.7.7.62)/GTP:AdoCbi-P guanylyltransferase (EC 3.1.3.73) and is required for de novo synthesis of AdoCbl (coenzyme B(12)). Here we show that ORF MJ1117 of the hyperthermophilic, methanogenic archaeon Methanocaldococcus jannaschii encodes a CobY protein (Mj CobY) that transfers the GMP moiety of GTP to AdoCbi-P to form AdoCbi-GDP. Results from isothermal titration calorimetry (ITC) experiments show that MjCobY binds GTP (K(d) = 5 muM), but it does not bind the GTP analogues GMP-PNP (guanosine 5'-(beta,gamma)-imidotriphosphate) or GMP-PCP (guanylyl 5'-(beta,gamma)-methylenediphosphonate) nor GDP. Results from ITC experiments indicate that MjCobY binds one GTP per dimer. Results from in vivo studies support the conclusion that the 5'-deoxyadenosyl upper ligand of AdoCbi-P is required for MjCobY function. Consistent with these findings, MjCobY displayed high affinity for AdoCbi-P (K(d) = 0.76 muM) but did not bind nonadenosylated Cbi-P. Kinetic parameters for theMj CobY reaction were determined. Results from circular dichroism studies indicate that, in isolation, MjCobY denatures at 80 degrees C with a concomitant loss of activity. We propose that ORF MJ1117 of M. jannaschii be annotated as cobY to reflect its involvement in AdoCbl biosynthesis.

Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii.

Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding, and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here, we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13 A pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.

Mechanism of substrate unfolding and translocation by the regulatory particle of the proteasome from Methanocaldococcus jannaschii.

In the archaebacterium Methanocaldococcus jannaschii (M. jannaschii), the proteasomal regulatory particle (RP), a homohexameric complex of proteasome-activating nucleotidase (PAN), is responsible for target protein recognition, followed by unfolding and translocation of the bound protein into the core particle (CP) for degradation. Guided by structure-based mutagenesis, we identify amino acids and structural motifs that are essential for PAN function. Key residues line the axial channel of PAN, defining the apparent pathway of substrate translocation. Subcomplex II of PAN, comprising the ATPase domain, associates with the CP and drives ATP-dependent unfolding of the substrate protein, whereas the distal subcomplex I forms the entry port of the substrate translocation channel. A linker segment between subcomplexes I and II is essential for PAN function, implying functional and perhaps mechanical coupling between these domains. Sequence conservation suggests that the principles of PAN function are likely to apply to the proteasomal RP of eukaryotes.

The crystal structure of C176A mutated [Fe]-hydrogenase suggests an acyl-iron ligation in the active site iron complex.

[Fe]-hydrogenase is one of three types of enzymes known to activate H(2). Crystal structure analysis recently revealed that its active site iron is ligated square-pyramidally by Cys176-sulfur, two CO, an "unknown" ligand and the sp(2)-hybridized nitrogen of a unique iron-guanylylpyridinol-cofactor. We report here on the structure of the C176A mutated enzyme crystallized in the presence of dithiothreitol (DTT). It suggests an iron center octahedrally coordinated by one DTT-sulfur and one DTT-oxygen, two CO, the 2-pyridinol's nitrogen and the 2-pyridinol's 6-formylmethyl group in an acyl-iron ligation. This result led to a re-interpretation of the iron ligation in the wild-type.

The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site.

Biological formation and consumption of molecular hydrogen (H2) are catalyzed by hydrogenases, of which three phylogenetically unrelated types are known: [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenase. We present a crystal structure of [Fe]-hydrogenase at 1.75 angstrom resolution, showing a mononuclear iron coordinated by the sulfur of cysteine 176, two carbon monoxide (CO) molecules, and the sp2-hybridized nitrogen of a 2-pyridinol compound with back-bonding properties similar to those of cyanide. The three-dimensional arrangement of the ligands is similar to that of thiolate, CO, and cyanide ligated to the low-spin iron in binuclear [NiFe]- and [FeFe]-hydrogenases, although the enzymes have evolved independently and the CO and cyanide ligands are not found in any other metalloenzyme. The related iron ligation pattern of hydrogenases exemplifies convergent evolution and presumably plays an essential role in H2 activation. This finding may stimulate the ongoing synthesis of catalysts that could substitute for platinum in applications such as fuel cells.

Divergence of selenocysteine tRNA recognition by archaeal and eukaryotic O-phosphoseryl-tRNASec kinase.

Selenocysteine (Sec) biosynthesis in archaea and eukaryotes requires three steps: serylation of tRNA(Sec) by seryl-tRNA synthetase (SerRS), phosphorylation of Ser-tRNA(Sec) by O-phosphoseryl-tRNA(Sec) kinase (PSTK), and conversion of O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) by Sep-tRNA:Sec-tRNA synthase (SepSecS) to Sec-tRNA(Sec). Although SerRS recognizes both tRNA(Sec) and tRNA(Ser) species, PSTK must discriminate Ser-tRNA(Sec) from Ser-tRNA(Ser). Based on a comparison of the sequences and secondary structures of archaeal tRNA(Sec) and tRNA(Ser), we introduced mutations into Methanococcus maripaludis tRNA(Sec) to investigate how Methanocaldococcus jannaschii PSTK distinguishes tRNA(Sec) from tRNA(Ser). Unlike eukaryotic PSTK, the archaeal enzyme was found to recognize the acceptor stem rather than the length and secondary structure of the D-stem. While the D-arm and T-loop provide minor identity elements, the acceptor stem base pairs G2-C71 and C3-G70 in tRNA(Sec) were crucial for discrimination from tRNA(Ser). Furthermore, the A5-U68 base pair in tRNA(Ser) has some antideterminant properties for PSTK. Transplantation of these identity elements into the tRNA(Ser)(UGA) scaffold resulted in phosphorylation of the chimeric Ser-tRNA. The chimera was able to stimulate the ATPase activity of PSTK albeit at a lower level than tRNA(Sec), whereas tRNA(Ser) did not. Additionally, the seryl moiety of Ser-tRNA(Sec) is not required for enzyme recognition, as PSTK efficiently phosphorylated Thr-tRNA(Sec).

Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation.

Selenocysteine (Sec)-decoding archaea and eukaryotes employ a unique route of Sec-tRNA(Sec) synthesis in which O-phosphoseryl-tRNA(Sec) kinase (PSTK) phosphorylates Ser-tRNA(Sec) to produce the O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) substrate that Sep-tRNA:Sec-tRNA synthase (SepSecS) converts to Sec-tRNA(Sec). This study presents a biochemical characterization of Methanocaldococcus jannaschii PSTK, including kinetics of Sep-tRNA(Sec) formation (K(m) for Ser-tRNA(Sec) of 40 nM and ATP of 2.6 mM). PSTK binds both Ser-tRNA(Sec) and tRNA(Sec) with high affinity (K(d) values of 53 nM and 39 nM, respectively). The ATPase activity of PSTK may be activated via an induced fit mechanism in which binding of tRNA(Sec) specifically stimulates hydrolysis. Albeit with lower activity than ATP, PSTK utilizes GTP, CTP, UTP and dATP as phosphate-donors. Homology with related kinases allowed prediction of the ATPase active site, comprised of phosphate-binding loop (P-loop), Walker B and RxxxR motifs. Gly14, Lys17, Ser18, Asp41, Arg116 and Arg120 mutations resulted in enzymes with decreased activity highlighting the importance of these conserved motifs in PSTK catalysis both in vivo and in vitro. Phylogenetic analysis of PSTK in the context of its 'DxTN' kinase family shows that PSTK co-evolved precisely with SepSecS and indicates the presence of a previously unidentified PSTK in Plasmodium species.

The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site.

Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1(ts)) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 A resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 A resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 A resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase.

Small-angle X-ray scattering reveals the solution structure of the peripheral stalk subunit H of the A1AO ATP synthase from Methanocaldococcus jannaschii and its binding to the catalytic A subunit.

The H subunit of the A1AO ATP synthase is a component of one of the peripheral stalks connecting the A1 and AO domain. Subunit H of the Methanocaldococcus jannaschii A1AO ATP synthase was analyzed by small-angle X-ray scattering (SAXS) in order to determine the first low-resolution structure of this molecule in solution. Independent to the concentration used, the protein is dimeric and has a boomerang-like shape, divided into two arms of 12.0 and 6.8 nm in length. Circular dichroism (CD) spectroscopy revealed that subunit H is comprised of 78% alpha-helix and a coiled-coil arrangement. To understand the orientation of the helices and the localization of the N- and C-termini inside the dimer, three truncated forms of subunit H (H8-104, H1-98, and H8-98) were expressed, purified, and analyzed by CD. SAXS experiments of H1-98 show that the maximum dimension of the truncated protein dropped to 15.1 nm. Comparison of the low-resolution shapes of H and H1-98 indicates that this goes along with structural changes in the C-terminal arm of the boomerang-like structure. Together with the result of a disulfide formation of a fourth truncated form, H1-47, with a cysteine at position 47, the data suggest a parallel alpha-helical interaction. In addition, all four truncated proteins are dimeric in solution. Tryptophan emission spectra showed specific binding of H and H8-104 to the neighboring, catalytic A subunit, which could not be detected in the presence of H1-98. Finally, the arrangement of H within the A1AO ATP synthase is presented.

Biosynthesis of phosphoserine in the Methanococcales.

Methanococcus maripaludis and Methanocaldococcus jannaschii produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA(Cys) with l-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in Methanococcales, cell extracts of M. maripaludis were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from M. maripaludis had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from M. jannaschii converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The M. maripaludis homolog complemented a serC mutation in the Escherichia coli phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.