Biosynthesis of Phosphoserine in the Methanococcales

Chemicals.PHP (hydroxypyruvic acid phosphate) was produced from the hydroxypyruvic acid dimethylketal cyclohexylammonium salt by acid hydrolysis according to the manufacturer's instructions (Sigma). The PHP concentration was determined by the analysis of inorganic phosphate in samples treated with bacterial alkaline phosphatase (Fermentas), correcting for inorganic phosphate in the unhydrolyzed sample (12). Sulfopyruvate was synthesized from bromopyruvate (49). Aqueous solutions of amino acids were prepared as potassium salts. All other chemicals were of the highest reagent grade available and were used without further purification.

Cloning of serA and aspC/serC genes.Genes were amplified from chromosomal DNA of Methanococcus maripaludis S2 or Methanocaldococcus jannaschii JAL-1 by using PCR. The M. maripaludis serA gene (MMP1588; GenBank accession no. NP_987511.1) was cloned between NheI and BamHI sites of the modified plasmid pET-15b (Novagen) to produce vector pGRDS30. The NheI-BamHI fragment containing MMP1588 was subcloned into pET-11a (Novagen) to produce vector pDG304. The M. maripaludis aspC/serC gene (MMP0391; GenBank accession no. NP_987511.1) was cloned between NdeI and XhoI sites of plasmid pET-20b (Novagen). The M. jannaschii aspC/serC gene (MJ0959; accession no. NC_000909) was cloned between NdeI and BamHI sites of plasmid pET-19b (Novagen) to produce vector pDG245. Plasmids were propagated in E. coli DH5α or DH10B (Invitrogen). The oligodeoxynucleotide primers used for PCR were MJ0959Fwd (5′-GCCATATGAAAATAGATGCAGTTAAAAAGC-3′), MJ0959Rev (5′-GCGGATCCTTATTCTTTCAATAGAACTTCTTTTGC-3′), M0391FN (5′-GATCCAAAGCATATGAAACAGATGGATACTGAAAAAC-3′), M0391RX (5′-CTTTGGATCCTCGAGATTTGATAGAACTTTTTTTGCAGC-3′), 5MMP1588N (5′-ATTCATTTAGGTGCTAGCATGTCAAAAATACTTATAACTGACCCGC-3′), and 3MMP1588B (5′-CCTGCCCCCGAAAAATTAGGATCCATTAAATTAGATGTT-3′). Dideoxyribonucleotide sequencing confirmed the sequences of inserts in recombinant plasmids.

Complementation of E. coli serC mutation.The MMP0391 gene was cloned into EcoRI and HindIII restriction sites of plasmid vector pKQV4 to express the gene under the control of a tac promoter (42). The recombinant plasmid was transformed into the serC mutant E. coli KL285 (CGSC 4310) (5). The pKQV4 plasmid was used as an empty vector control, and the E. coli serC gene cloned into pKQV4 was used as a positive control in complementation experiments. A single colony of each transformant was picked from LB agar plates containing ampicillin and grown in liquid M9 minimal medium containing 0.2% (wt/vol) d-glucose and amino acids. Amino acids were used at a concentration of 10 μg ml⁻¹, except for serine, which was used at 50 μg ml⁻¹. All media contained 100 μg ml⁻¹ ampicillin. Plates were grown for 36 h at 30°C. Digital images of plates were inverted and transformed using Photoshop software (Adobe) to enhance contrast.

Protein expression and purification. E. coli BL21(DE3) (Novagen) transformed with expression vectors was grown in Luria broth containing ampicillin (100 μg ml⁻¹) at 37°C with shaking at 250 rpm. When cultures reached an optical density at 600 nm (OD₆₀₀) of 0.6 to 0.7, protein expression was induced by the addition of α-d-lactose (1% [wt/vol]). After 3 hours of incubation with the inducer, cells were harvested by centrifugation and stored at −20°C.

Protein purification.Polyhistidine-tagged proteins were purified and separated from native E. coli proteins by Ni²⁺-affinity chromatography. E. coli cells containing heterologously expressed protein were suspended in cold binding buffer containing 20 mM sodium phosphate (pH 7.4) and 0.5 M NaCl. Cells were lysed by passage through a French pressure mini cell at 8,000 lb/in² (Thermo Electron) and then sonicated on ice for 2 min, using a Sonifier 450 with a microtip (15 W, 30% duty) (Branson) to reduce viscosity. Lysates were clarified by centrifugation (15,000 × g for 10 min at 4°C), and the cell extract was applied to a 5-ml HisTrap column (GE Healthcare) equilibrated with binding buffer. Chromatography was performed using an ÄKTAprime system (GE Healthcare) at a flow rate of 5 ml min⁻¹. Protein was eluted from the column with a linear gradient to 100% elution buffer over 20 min. Elution buffer contained 20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, and 0.5 M imidazole. Fractions containing the target protein were identified by their absorbance at 280 nm, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and activity assay.

Fractions containing the His₆-MMP1588 protein were pooled and concentrated in a stirred ultrafiltration cell (Amicon) with a 10-kDa-molecular-size cutoff filter (Pall) under N₂. The retentate was desalted using a 5-ml HiTrap Sepharose G-25 column (GE Healthcare) equilibrated in 50 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-KOH (pH 7.5). Fractions containing the His₁₀-MJ0959 protein were dialyzed at 4°C against buffer containing 50 mM HEPES-KOH (pH 7.4) and 5 mM MgCl₂. Protein was concentrated inside dialysis tubing, using polyethylene glycol.

Small-scale purifications were carried out using spin columns containing Ni-Sepharose resin (QIAGEN). Cell extract was applied to columns equilibrated with binding buffer, followed by centrifugation (200 × g for 1 min at room temperature). Columns were washed with binding buffer, and protein was eluted with 200 μl elution buffer. Total protein concentration was determined using the Bio-Rad protein assay or the BCA assay (Pierce), with bovine serum albumin (fraction V) as a standard.

Cell extracts were prepared from 85 mg M. maripaludis 900 cells grown in minimal medium under a H₂-CO₂ atmosphere in sealed serum vials at 37°C (29, 30). Cells were suspended in 1 ml aerobic buffer containing 50 mM TES-NaOH and 10 mM MgCl₂ (pH 7.2) and lysed by sonication. Lysates were clarified by centrifugation (18,000 × g for 10 min at 4°C), and the soluble cell extract was stored at −20°C.

Analytical size exclusion chromatography.Native masses of purified proteins were measured by size exclusion chromatography. Protein was applied to a BioSep-SEC-S 3000 column (7.8 mm by 300 mm by 5 μm; Phenomenex) with a Security Guard SEC cartridge (4 by 3 mm; Phenomenex) that was equilibrated in aqueous 50 mM sodium phosphate buffer (pH 6.8). High-performance liquid chromatography (HPLC) was performed at a flow rate of 1.0 ml min⁻¹ at ambient temperature, using an isocratic elution program with starting buffer. Proteins were detected by their absorbance values at 220 and 280 nm, using a System Gold 168 photodiode array detector (Beckman). Standards used to calibrate the column were apoferritin, β-amylase, aldolase, conalbumin, carbonic anhydrase, myoglobin, cytochrome c, and adenosyl cobalamine (Sigma).

Oxidoreductase enzyme activity assays.Oxidation rates of d-3-phosphoglycerate were calculated from the corresponding amounts of NADH produced (52). Standard reaction mixtures (1 ml) contained 100 mM Tris, 100 mM hydrazine sulfate, 2 mM dithiothreitol (DTT), 1 mM d-3-phosphoglycerate, 1 mM β-NAD⁺, and 1 mM EDTA, adjusted to pH 8.5 with NaOH. Reaction mixtures were preincubated at 37°C, and reactions were initiated by the addition of phosphoglycerate. Reduction of PHP was similarly measured in standard reaction mixtures containing 100 mM morpholineethanesulfonic acid (MES)-KOH (pH 6.0), 5 mM EDTA, 150 μM NADH, 100 mM KCl, and 82 μM PHP.

A DU-800 spectrophotometer (Beckman) was used to measure NADH absorption at 340 nm, assuming a molar absorptivity of 6,317 M⁻¹ cm⁻¹ for NADH at 25°C (24). Reactions were performed in optical glass cuvettes (Starna) at 37°C with a Peltier temperature-controlled sampler (Beckman). Initial rates were estimated by linear regression, using the linear portion of the reaction progress curve. One unit of enzymatic activity catalyzed the reduction of 1 μmol NAD⁺ per min in a 1-ml reaction mix. Kinetic constants were estimated by nonlinear regression of initial rate data using the KaleidaGraph program (Synergy Software).

Aminotransferase enzyme activity assays.Aminotransferase activity was detected by precolumn derivatization and chromatographic separation with fluorescence detection (20). Standard reaction mixtures (100 μl) contained 50 mM HEPES-KOH (pH 7.5), 80 mM KCl, 10 mM MgCl₂, 2 mM amino acids, 2 mM 2-oxoacid, and 100 μM pyridoxal 5′-phosphate (PLP). Primary amines in a portion of the reaction product were derivatized in a 100-μl reaction mix containing 35% (vol/vol) acetonitrile, 0.1 M sodium borate (pH 9), 1 mM naphthalene-2,3-dicarboxaldehyde, and 5 mM sodium cyanide. Reaction mixtures were incubated in the dark at 25°C for 1 h and then diluted with the HPLC mobile phase.

Reversed-phase HPLC was performed to separate the fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives. Amino acid CBI derivatives were applied to a reversed-phase C₁₈ column (4.6 mm by 250 mm by 5 μm; Axxium) with a Security Guard ODS cartridge (4 by 3 mm; Phenomenex) that was equilibrated in the mobile phase, containing 50% (vol/vol) acetonitrile, 15 mM phosphoric acid, 10 mM triethylamine, and water. Isocratic elution with this mobile phase was used at a flow rate of 1 ml min⁻¹ at 35°C. Samples (25 μl) were injected using a Beckman 508 autosampler. An FP-2020+ fluorescence detector (Jasco) was used with an excitation wavelength of 420 nm and an emission wavelength of 488 nm. Data were collected and chromatograms were integrated using 32 Karat software (Beckman). Product formation was calculated from the integrated peak area of the chromatogram, using external standards for calibration. One unit of aminotransferase activity catalyzed the transfer of amino groups from 1 μmol substrate per min in a 100-μl reaction mix. CBI derivatives of pure amino acids were used as external standards. Under these conditions, retention factors for the CBI derivatives were 0.16 (l-cysteate), 0.18 (dl-phosphoserine), 0.50 (l-glutamine), 0.53 (l-aspartate), 0.61 (l-serine), 0.67 (l-glutamate), 1.11 (glycine), 1.37 (l-cysteine), and 1.48 (l-alanine).

Amino acids were derivatized with ethylchloroformate and analyzed by gas chromatography-mass spectrometry (GC-MS). Reaction products were mixed with ethylchloroformate in a solution of water-trifluoroethanol-pyridine (60:32:8 [vol/vol/vol]) (46). Derivatives were analyzed on a Finnigan MAT GCQ GC-MS with a DB-5MS capillary column (0.32 mm × 29 m, with a 0.5-μm film; J&W Scientific). The injector temperature was 270°C, and the initial column temperature was 80°C. After a 2-min delay, the temperature was increased to 275°C over 5 min, with a column ramp temperature of 40°C min⁻¹. Methane was used for chemical ionization in the positive mode, and a mass range of 100 to 600 units was scanned, with an approximate scan time of 0.59 s. Using this method, the N-ethoxycarbonyl trifluoroethyl ester derivatives had the following retention times and mass spectral data. The molecular ion (MH⁺) is shown first, if observed, followed by the base peak (in italics), and characteristic fragment ions are listed in decreasing order of intensity: 2-oxoglutarate, 4.51 min (311, 183, 211, and 291) and 5.45 min (183 and 211); alanine, 4.59 min (244, 116, 170, 224, and 144); phosphoserine, 4.62 min (242, 142, 222, 170, and 114); aspartate, 5.62 min (370, 242, 270, and 350); and glutamate, 6.04 min (284 and 256).

Kinetics of oxaloacetate decarboxylation.Rates of oxaloacetate decarboxylation were measured by UV absorbance spectroscopy (22). Reaction mixtures contained 0.3 to 2.3 mM freshly prepared oxaloacetate added to 300 μl of solution containing 50 mM HEPES-KOH (pH 7.5) and 80 mM KCl at 50°C. Absorbance of the oxaloacetate enolic tautomer was measured at 260 nm in a quartz cuvette at 50°C. The rate constant was calculated by linear regression of the measured initial rates at various oxaloacetate concentrations.

Phylogeny of euryarchaeal aspartate aminotransferases.An alignment of 31 aminotransferase homologs was prepared using the T-Coffee program (v. 3.27) (32). The phylogeny was inferred using a Bayesian inference method implemented by the MrBayes program (v. 3.1.2) (35), with four chains, the default mixed model of amino acid replacements with fixed-rate matrices, and a γ-distribution of rates approximated with four categories. Alternative trees were produced using the proml program or the protdist and neighbor programs (with 100 bootstrap replicates) (10). Both programs used the Jones-Taylor-Thornton model of amino acid changes and assumed a γ-distribution of rates (α = 2.4) approximated by three states.

M. maripaludis uses the phosphorylated pathway for serine biosynthesis.To establish that M. maripaludis expresses enzymes required for the phosphorylated pathway of serine biosynthesis, enzyme activities were measured in cell extracts. Reaction mixtures containing cell extract, 123 μM PHP, and 150 μM NADH produced d-3-phosphoglycerate and NAD⁺, with a specific activity of 11 mU mg⁻¹ protein. Reaction mixtures containing extract, 40 μM PHP, and 5 mM l-aspartate or l-glutamate completely converted PHP to phosphoserine (Fig. 2). These phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities confirmed the presence of the phosphorylated pathway to produce phosphoserine and serine in M. maripaludis. Similar activities were detected in extracts from M. jannaschii. Alternatively, these methanogens could phosphorylate serine directly to produce phosphoserine if they express a serine kinase enzyme. A cell extract of M. maripaludis (44 μg) was incubated with 10 mM l-serine and 5 mM Mg-ATP for 30 min at 30°C in a 100-μl reaction mix to test for serine kinase activity. No phosphoserine was detected in the reaction product (<2 μM).

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FIG. 2.

Cell extract of M. maripaludis catalyzes the transamination of aspartate and phosphohydroxypyruvate to produce phosphoserine. Reaction mixtures containing 40 μM PHP, 5 mM l-aspartate, and 44 μg M. maripaludis cell extract were incubated for 30 min at 30°C. The CBI derivatives of the amino acids were separated by reversed-phase HPLC and detected by their fluorescence. All of the PHP in the reaction mix was converted to phosphoserine, based on comparison of the phosphoserine-CBI peak area to an external standard. The aspartate-CBI peak is off scale in these overlaid chromatograms.

Characterization of M. maripaludis 3-phosphoglycerate dehydrogenase.The M. maripaludis PGDH (MMP1588) amino acid sequence is similar to those of bacterial and eukaryotic homologs. These proteins share a carboxy-terminal ACT domain (an amino acid allosteric binding site) that is not found in other members of the 2-hydroxyacid dehydrogenase family (2). Residues identified in the active site of E. coli PGDH are highly conserved in methanogenic orthologs, except for Asn¹⁰⁸, which is replaced with serine in the nicotinamide binding site (39). A homology model of the MMP1588 protein was produced by the SWISS-MODEL server, based on PGDH crystal structure models deposited in the Protein Data Bank (40). The model included no significant clashes, emphasizing the high degree of conservation in this protein.

To test the predicted function of MMP1588 in phosphoserine biosynthesis, we cloned the gene and expressed the protein, fused to an amino-terminal hexahistidine tag, heterologously in E. coli. Purified His₆-MMP1588 had an apparent mass of 63 kDa, as determined by SDS-polyacrylamide gel electrophoresis (Fig. 3). This value is close to the protein's expected mass of 59 kDa. Analytical size exclusion chromatography identified large aggregates of protein that eluted in the column void volume as well as smaller forms with Stokes radii of 49 and 35 Å, corresponding to tetrameric and dimeric forms of the protein, respectively.

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FIG. 3.

His₆-MMP1588 (PGDH) and His₁₀-MJ0959 (AspAT) were purified to homogeneity by affinity chromatography. Proteins were separated by SDS-PAGE and stained with Coomassie blue dye. Lanes M, broad-range protein standards (Bio-Rad), with masses indicated in kDa; lane 1, purified His₆-MMP1588; lane 2, purified His₁₀-MJ0959.

The purified enzyme catalyzed the NAD⁺-dependent oxidation of d-3-phosphoglycerate, with a specific activity of 55 mU mg⁻¹ protein, corresponding to a rate of 3.2 min⁻¹. No significant activity was detected using NADP⁺ instead of NAD⁺ (<0.005 U mg⁻¹). The enzyme had maximal catalytic activity at 45°C, with no activity observed at 60°C. However, preincubation of the protein at 45°C for 15 min reduced its activity by 50%, while it retained 80% activity after 30 min of incubation at 37°C. Therefore, standard assays were performed at 37°C. The His₆-MMP1588 enzyme did not require Mg²⁺ or thiol reductants for full activity: neither 20 mM MgCl₂ nor 1 mM DTT stimulated activity.

No NAD⁺ reduction was observed using 1 mM d-malate, dl-lactate, glycolate, dl-glycerate, or 2-hydroxyisocaproate as an alternative substrate, nor did these 2-hydroxyacids inhibit the oxidation of 1 mM d-3-phosphoglycerate. In reaction mixtures containing various concentrations of d-3-phosphoglycerate and 0.2 mM NAD⁺, the enzyme had an apparent K_m equal to 370 ± 110 μM and a turnover number of 3.7 ± 0.29 min⁻¹. No inhibition was detected in reaction mixtures containing 1 to 3 mM l-serine.

Operating in the reverse direction, His₆-MMP1588 catalyzed the reduction of PHP, with a specific activity of 34 U mg⁻¹ protein (33 s⁻¹). No significant activity was detected with 2-oxoglutarate, oxaloacetate, pyruvate, glyoxylate, or formate in place of PHP (<0.03 U mg⁻¹). However, the enzyme did reduce sulfopyruvate, a PHP analog, with a specific activity of 0.1 U mg⁻¹. His₆-MMP1588 had optimal catalytic activity (reducing PHP) at pH 5.3 but retained 75% activity from pH 4.5 to 9. Potassium chloride stimulated activity, as reaction mixtures containing 100 mM KCl produced twofold more NAD⁺ than did reaction mixtures without added salt. Apparent kinetic parameters for PHP reduction were as follows: K_M , 49 ± 5.3 μM; and k _cat, 47 ± 2.4 s⁻¹.

The ACT domains of several bacterial d-3-phosphoglycerate dehydrogenases bind the effector l-serine cooperatively, causing allosteric inhibition (39). For example, Mycobacterium tuberculosis PGDH has an I _0.5 of 30 μM, and E. coli PGDH has an I _0.5 of 2 to 4 μM l-serine (7), where I _0.5 is the inhibitor concentration that causes 50% maximal activity. Although M. maripaludis PGDH contains a carboxy-terminal ACT domain, it is poorly inhibited by l-serine. Preincubation with 1 to 3 mM l-serine reduced the rate of PHP reduction by 27 to 46%. Similar incubations with 3 mM l-glutamate reduced activity by 38%. d-Serine, l-alanine, dl-phosphoserine, dl-cysteine, and 2-oxoglutarate did not significantly inhibit PHP reduction at a 3 mM concentration. Bacillus subtilis PGDH becomes desensitized to serine inhibition during incubation without DTT (37). Therefore, we incubated M. maripaludis PGDH with 20 mM DTT and 10 mM EDTA at 4°C for 24 h. The DTT-treated enzyme was no more sensitive to inhibition: l-serine (1 to 3 mM) inhibited PHP reduction by 43 to 51%.

Although the polyhistidine tag on the MMP1588 protein enabled facile separation of the heterologous protein from the native E. coli PGDH, it is possible that the tag could interfere with protein subunit assembly and the formation of the regulatory domain. Therefore, the MMP1588 protein was expressed without a fusion tag in E. coli BL21(DE3)/pDG304. SDS-PAGE analysis of a cell extract from that lactose-induced strain showed a soluble protein corresponding to MMP1588, which had an apparent mass of 55 kDa and accounted for 30% of the total Coomassie blue-stained protein. The extract had a specific activity of 15 U mg⁻¹ in standard PGDH activity assays measuring the NADH-dependent reduction of PHP. In the presence of 3 mM l-serine, the extract had a specific activity of 12 U mg⁻¹, while reaction mixtures containing 3 mM l-cysteine or dl-phosphoserine demonstrated somewhat lower activities, of 10 and 8.5 U mg⁻¹, respectively. The native E. coli PGDH did not contribute significantly to this activity, as a cell extract of E. coli BL21(DE3)/pET-11a contained <0.25 U mg⁻¹ activity (the background rate of PHP-independent NADH oxidation). Therefore, the presence of an amino-terminal polyhistidine tag does not account for the insensitivity of M. maripaludis PGDH to allosteric inhibition by serine.

Broad-specificity aspartate aminotransferase catalyzes phosphoserine production.The M. maripaludis MMP0391 protein was annotated as an aspartate aminotransferase (AspAT) by the genome sequencing project (17). Because a homologous AspAT from Methanothermobacter thermautotrophicus was reported to catalyze the transamination of phosphoserine (43, 44), we proposed that a single enzyme could catalyze both reactions in the Methanococcales. The MMP0391 gene was cloned into plasmid pKQV4 downstream from a tac promoter. In the presence of the isopropyl-β-d-thiogalactopyranoside (IPTG) inducer, this gene complements the serC mutation of E. coli KL285 (5) (Fig. 4). The E. coli KL285/pKQV4 control strain did not grow on serine-free medium. Slight growth of E. coli KL285/pKQV4(MMP0391) in the absence of IPTG may be caused by leaky expression from the tac promoter.

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FIG. 4.

The M. maripaludis MMP0391 gene complements the serC mutation of E. coli KL285. (A) M9 minimal medium with glucose containing all 20 amino acids supports the growth of E. coli KL285 containing (clockwise from top) pKQV4 (E. coli serC), empty pKQV4 vector, and pKQV4 (MMP0391). (B) Minimal medium containing 19 amino acids (without serine). (C) Minimal medium containing 19 amino acids (without serine) and 1 mM IPTG. All media contain 100 μg ml⁻¹ ampicillin.

The MMP0391 protein was fused to a carboxy-terminal polyhistidine tag and expressed in E. coli. The heterologously expressed protein had an apparent mass of 44.3 kDa, similar to the expected mass of 42.9 kDa. However, most of the expressed protein was found in the insoluble portion of cell lysate. Protein solubilized from inclusion bodies had no detectable aminotransferase activity (data not shown). Therefore, the orthologous protein from M. jannaschii, MJ0959, was chosen for biochemical characterization.

The MJ0959 protein is 70% identical to the MMP0391 protein, and 91% of the aligned amino acid residues are similar. The His₁₀-MJ0959 protein was expressed heterologously as a soluble protein with an apparent mass of 45.4 kDa, which is close to its predicted mass of 45.2 kDa. The protein was purified by Ni²⁺-affinity chromatography and desalted by dialysis. From 250 ml of E. coli expressing the heterologous protein, we obtained 17 mg of purified protein. This protein preparation was at least 95% pure, as judged by SDS-polyacrylamide gel electrophoresis (Fig. 3) and analytical size exclusion chromatography. The protein was stable during heating at 70°C for 10 min.

Analytical size exclusion chromatography showed that the protein forms a high-molecular-weight complex with a 57-Å Stokes radius, which corresponds to a 330,000-Da apparent mass. Therefore the protein probably forms an octameric complex. No peaks corresponding to tetrameric, dimeric, or monomeric proteins were observed. The high-molecular-weight species had absorbance maxima at 278, 331, and 416 nm, consistent with a PLP internal aldimine (45). Based on published molar absorption coefficients for PLP enzymes, 70% of the purified MJ0959 protein is bound to PLP (27).

Purified MJ0959 catalyzed the transamination of 2-oxoglutarate from l-aspartate to produce glutamate and oxaloacetate. Glutamate was identified by GC-MS, as the N-ethoxycarbonyl trifluoroethyl ester, and by HPLC, as the fluorescent CBI derivative. The enzyme catalyzed glutamate formation with a specific activity of 19 U mg⁻¹, corresponding to a rate of 14 s⁻¹. In the reverse reaction, the transamination of oxaloacetate from l-glutamate, the enzyme had a lower specific activity, i.e., 0.3 U mg⁻¹ (0.2 s⁻¹). After a 2-h incubation with excess enzyme (11 μg) at 50°C, 5 mM l-aspartate and 5 mM 2-oxoglutarate were converted to 2.6 mM glutamate and 2.2 mM alanine. Alanine formation was confirmed by GC-MS and coelution of an alanine-CBI derivative during HPLC. A reaction mix containing enzyme and 5 mM aspartate with no 2-oxoacid produced only 0.2 mM alanine. Therefore, alanine is probably formed through the enzyme-catalyzed transamination of pyruvate produced by the spontaneous decomposition of oxaloacetate. The rate of nonenzymatic oxaloacetate decarboxylation was measured under similar conditions, and the reaction was found to be of the first order with respect to oxaloacetate, with a rate constant of 2.5 × 10⁻⁴ s⁻¹. This measured rate constant is similar to those reported previously for uncatalyzed oxaloacetate decarboxylation (11), and it is sufficiently high to account for the observed alanine. However, we cannot rule out alanine formation as a β-decarboxylation side reaction catalyzed by MJ0959 without further experiments.

This enzyme also catalyzed the transamination of PHP, using either l-aspartate or l-glutamate as an amino group donor (Fig. 5). In reaction mixtures containing 5 mM l-aspartate and 1.2 mM PHP, the enzyme catalyzed phosphoserine production, with a specific activity of 1.5 U mg⁻¹ (1.1 s⁻¹). A small amount of alanine was produced in this reaction as well. l-Glutamate could also serve as an amino group donor for the transamination of PHP, and no alanine was detected in these reaction mixtures. The reverse reaction, using phosphoserine as a donor, produced aspartate and glutamate from oxaloacetate and 2-oxoglutarate, respectively.

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FIG. 5.

Purified MJ0959 protein catalyzes the transamination of aspartate and phosphohydroxypyruvate to produce phosphoserine and alanine. Reaction mixtures were incubated at 50°C. Fluorescent CBI derivatives were separated and analyzed by HPLC as described in the legend to Fig. 2.

Sulfopyruvate is an analog of oxaloacetate and an intermediate in methanogenic coenzyme M biosynthesis (14). When incubated with aspartate or glutamate in enzymatic reaction mixtures, sulfopyruvate was transaminated to form cysteate. Reaction mixtures containing oxaloacetate or 2-oxoglutarate and l-cysteate acid produced aspartate and glutamate, respectively. Other 2-oxoacid substrates for the purified enzyme included glyoxylate and pyruvate. However, glycine, l-alanine, and l-serine were poor amino group donors for the reverse reaction.

Vertical inheritance of class V aspartate aminotransferases in methanogenic archaea.All of the methanogenic euryarchaea have orthologs of the archaeal aspartate aminotransferase (Fig. 6). Phylogenetic analysis showed that this gene was vertically inherited among the euryarchaea, even the heterotrophic archaea Archaeoglobus fulgidus and Halobacterium sp., which probably evolved from a methanogenic ancestor. In contrast, the deeply diverging heterotrophic euryarchaea, such as Pyrococcus spp. and Thermoplasma spp., have no close homolog. The Thermococcales do have homologs of an uncharacterized aminotransferase family that includes crenarchaeal homologs, 2-aminoethylphosphonate aminotransferase from Salmonella enterica serovar Typhimurium, and human serine-pyruvate aminotransferase. Finally, several clostridia and most cyanobacteria also contain uncharacterized homologs that were likely acquired by horizontal gene transfer and then vertically inherited within modern bacterial lineages. The last group includes plant peroxisomal and α-proteobacterial serine-glyoxylate aminotransferases.

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FIG. 6.

Phylogeny of class V aspartate aminotransferase homologs constructed using a Bayesian inference method. The branches are labeled with organism names and the GenBank or SwissProt accession number for each sequence. The tree is arbitrarily rooted. Bar, 0.1 amino acid change expected per site. Numbers near each interior branch are clade credibility values, or the fraction of trees that contain each cluster of sequences shown in the consensus tree. This tree is generally congruent with trees produced using protein maximum likelihood and neighbor-joining distance methods. Branches with high clade credibility values also have high bootstrap values (from 100 neighbor-joining distance trees). Previously characterized enzymes are labeled AEPT (2-aminoethylphosphonate transaminase), SPT (serine-pyruvate transaminase), AGAT (alanine-glyoxylate aminotransferase), and SGAT (serine-glyoxylate aminotransferase).