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Journal of Bacteriology, January 2007, p. 575-582, Vol. 189, No. 2
0021-9193/07/$08.00+0     doi:10.1128/JB.01269-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biosynthesis of Phosphoserine in the Methanococcales{triangledown}

Sunna Helgadóttir,1 Guillermina Rosas-Sandoval,1 Dieter Söll,1 and David E. Graham2*

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520,1 Department of Chemistry and Biochemistry and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 787122

Received 11 August 2006/ Accepted 16 October 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Methanococcus maripaludis and Methanocaldococcus jannaschii produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNACys 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Many methanogenic archaea have a remarkable tRNA-dependent pathway for cysteine biosynthesis. In these organisms, an unusual class II aminoacyl-tRNA synthetase catalyzes the aminoacylation of tRNACys with L-phosphoserine (Sep) (38). This Sep-tRNACys is then sulfurylated to produce Cys-tRNACys for protein biosynthesis. This tRNA-dependent pathway for cysteine biosynthesis conserves the energy of a phosphoester bond compared to the canonical pathway, where phosphoserine is hydrolyzed to produce serine that is acetylated and then converted to cysteine by a sulfhydrolase. Methanocaldococcus jannaschii also uses phosphoserine to produce cystathionine (50). This paper addresses the biosynthesis of phosphoserine in Methanococcus maripaludis and M. jannaschii to decipher the relationship between cysteine and serine biosynthesis in the euryarchaea.

Three pathways for the biosynthesis of serine are the phosphorylated pathway, which produces a phosphoserine intermediate (16, 18), the nonphosphorylated pathway (36), and the serine pathway for incorporating formaldehyde by the reverse activity of serine hydroxymethyltransferase (34). Most microorganisms use the phosphorylated pathway, which requires three dedicated enzymes, namely, phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase, and phosphoserine phosphatase. Many eukaryotes and some bacteria employ the nonphosphorylated pathway, which requires phosphoglycerate kinase, glycerate dehydrogenase, and serine-pyruvate aminotransferase. The serine pathway for C1 assimilation is commonly found in methanotrophic and methylotrophic bacteria.

Isotopic labeling studies using nuclear magnetic resonance showed that serine is produced from pyruvate in the methanogens Methanococcus voltae (8), Methanospirillum hungatei (9), and Methanosphaera stadtmanae (28). These results demonstrated that serine hydroxymethyltransferase is not a significant source of serine for these methanogens, but these experiments could not distinguish between the phosphorylated and nonphosphorylated serine biosynthetic pathways. The metabolic reconstruction of M. jannaschii from its genome sequence predicted that methanogens use the phosphorylated pathway, based on the discovery of phosphoglycerate dehydrogenase and phosphoserine phosphatase homologs (4, 41).

PGDH (EC 1.1.1.95) catalyzes the NAD+-dependent oxidation of D-3-phosphoglycerate, forming 3-phosphohydroxypyruvate (PHP) (Fig. 1). This oxidoreductase belongs to a family of NAD(P)+-dependent formate, glycerate, and erythro-4-phosphate dehydrogenases (15). The M. maripaludis genome encodes two members of this family (MMP1588 and MMP0870), and members of the Thermococcales have three homologs. Because these homologs could catalyze different reactions, it is important to establish their functions, as no archaeal PGDH protein has been characterized biochemically. Although PGDH proteins from diverse organisms share a high degree of sequence similarity in their catalytic domains, they differ in their regulatory domains. In Escherichia coli and other bacteria, L-serine binds to a carboxy-terminal ACT domain of PGDH, allosterically regulating catalytic activity (39). While this feedback inhibition efficiently regulates serine production in many bacteria, it could conflict with separate phosphoserine requirements for cysteine and cystathionine biosynthesis in methanogens.


Figure 1
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  		FIG. 1. Biosynthesis of L-phosphoserine in the Methanococcales. PGDH catalyzes the oxidation of D-3-phosphoglycerate to produce phosphohydroxypyruvate, concomitant with the reduction of NAD+. A broad-specificity aminotransferase (AspAT) catalyzes the transamination reaction from L-glutamate to produce L-3-phosphoserine and {alpha}-ketoglutarate. Phosphoserine phosphatase produces L-serine for protein synthesis and glycine production. In a reaction with homocysteine (Hcy), phosphoserine produces cystathionine (50). Alternatively, Sep can be used to aminoacylate tRNACys, which can be converted to Cys-tRNACys by a sulfide transferase enzyme (38).

 
In E. coli and the archaeon Methanosarcina barkeri (26), a canonical phosphoserine transaminase (EC 2.6.1.52) catalyzes amino group transfer from L-glutamate to phosphohydroxypyruvate to produce L-phosphoserine. However, no ortholog of the M. barkeri serC gene was found in the genome of M. maripaludis or M. jannaschii (4, 17). Therefore, this reaction has been a missing step in metabolic reconstructions (4, 41). Phosphoserine phosphatase (EC 3.1.3.3) is conserved in all methanogen genomes, and the M. jannaschii protein has been characterized biochemically and crystallized (47).

We demonstrated that M. maripaludis contains phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities through in vitro assays. Heterologously expressed and purified phosphoglycerate dehydrogenase from M. maripaludis catalyzed the reversible formation of PHP but was poorly inhibited by L-serine. In the second step, a class V aspartate aminotransferase (AspAT) gene from M. maripaludis complemented a serC mutation in E. coli, permitting growth without exogenous serine. A homologous AspAT gene from M. jannaschii was heterologously expressed at high levels, and the purified protein catalyzed the transamination of aspartate, glutamate, phosphoserine, cysteate, and alanine. Together, these results confirm the prediction that methanogens use a phosphorylated serine pathway, and they suggest that the route to phosphoserine for tRNA-dependent cysteine biosynthesis evolved early in the euryarchaeal lineage.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 DH5{alpha} 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 {alpha}-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 x 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 x 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 x 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, ß-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 ß-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 x 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 {gamma}-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 {gamma}-distribution of rates ({alpha} = 2.4) approximated by three states.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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).


Figure 2
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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.


Figure 3
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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 Km 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: KM, 49 ± 5.3 µM; and kcat, 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 I0.5 of 30 µM, and E. coli PGDH has an I0.5 of 2 to 4 µM L-serine (7), where I0.5 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-ß-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.


Figure 4
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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 x 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 ß-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.


Figure 5
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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 {alpha}-proteobacterial serine-glyoxylate aminotransferases.


Figure 6
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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).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The M. maripaludis phosphoglycerate dehydrogenase has similar structural and kinetic properties to those of the well-characterized E. coli and M. tuberculosis PGDH proteins (Table 1). The M. tuberculosis protein has been called the link between E. coli and mammalian PGDHs, due to its extended carboxy-terminal domain, substrate inhibition kinetics, substrate specificity, and ionic strength requirements (7). M. maripaludis PGDH has a carboxy-terminal extension similar to that of M. tuberculosis PGDH and requires 100 to 200 mM KCl for maximal activity. Although gel filtration experiments did not resolve the size of native M. maripaludis PGDH, the results suggest that it forms a very-high-molecular-weight complex that can dissociate into tetrameric and dimeric forms.


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  		TABLE 1. Structural and kinetic properties of D-3-phosphoglycerate dehydrogenases

 
However, M. maripaludis PGDH is more similar to the B. subtilis and mammalian enzymes in its low sensitivity to inhibition by serine. The structural basis for this discrepancy remains to be determined, but the regulatory properties are consistent with the methanogen's requirement for phosphoserine to make cysteine and cystathionine. In E. coli, cysteine is made from free serine, so phosphoserine levels need only be controlled by the serine concentration. This linear pathway is well regulated through allosteric inhibition by the end product. In the Methanococcales, phosphoserine appears to be a branch point (Fig. 1). It is required for serine, cysteine, and cystathionine biosynthesis, so canonical feedback regulation of phosphoglycerate dehydrogenase by serine would decrease cysteine and cystathionine production, regardless of their levels. Similar branch points, such as the aspartokinase reaction, are regulated either by isozymes or by allosteric inhibition of downstream enzymes. The mode of regulation of these pathways is currently unknown for the Methanococcales.

Based on their structural similarity, the euryarchaeal aspartate aminotransferases belong to the large superfamily of PLP-dependent transferases and the cystathionine synthase-like family of proteins (31). These aspartate aminotransferases have been classified as either subgroup IV aminotransferases (25, 43) or class V aminotransferases (33). This classification distinguishes them from the canonical bacterial/eucaryal aspartate aminotransferases, which belong to subgroup I or class I, and reflects their separate evolutionary history. However, these classifications are not sufficient to predict the catalytic activities and physiological roles of the enzymes (13, 41). A recent study used o-phthalaldehyde derivatization and HPLC separation with fluorescence detection to study the activities of 20 aminotransferases from Corynebacterium glutamicum (23). Affinity purification of the heterologously expressed proteins reduced the possibility of contamination by other aminotransferases. This high-throughput, generic approach offers an unprecedented amount of information about an organism's aminotransferase complement but cannot screen all possible substrate combinations. We used analogous derivatization chemistry to characterize the class V AspAT from M. jannaschii.

The broad-specificity AspAT from M. jannaschii is an ortholog of the AspAT that was previously purified, cloned, and characterized from Methanothermobacter thermautotrophicus SF-4 (formerly Methanobacterium thermoformicicum) (43, 44). These proteins are probably orthologs of the AspAT purified from Methanococcus aeolicus (53). All three proteins formed high-molecular-weight complexes, which were probably tetramers and octamers. While these enzymes have the highest specific activities for catalyzing the transamination of aspartate and 2-oxoglutarate, they also have appreciable activities with alanine and phosphoserine. We showed that M. maripaludis AspAT complemented a serC mutation in E. coli; it remains to be determined whether AspAT is also the primary alanine aminotransferase in these methanogens.

Our finding that AspAT catalyzes cysteate transamination may be important for understanding the biosynthesis of coenzyme M in the Methanosarcinales. These methanogens have no homologs of the first three enzymes identified in the coenzyme M biosynthetic pathway of M. jannaschii but do have a functional sulfopyruvate decarboxylase enzyme (S. Namboori and D. E. Graham, unpublished data). Therefore, they must have another means to produce sulfopyruvate, such as the transamination of cysteate. Additional studies are required to determine the relevance of this pathway and the mechanism of cysteate formation in these strict anaerobes.

Separate archaeal lineages have evolved at least three different aspartate aminotransferases. The class V aminotransferases evolved in the euryarchaea after the divergence of the Thermococcales and Thermoplasmatales. Members of the Methanosarcinales also have homologs of the Thermus thermophilus bacterial subgroup IV aspartate aminotransferase (19). Although the Thermococcales and crenarchaea have homologs of the subgroup IV aminotransferases, the functions of these proteins are not known. Pyrococcus furiosus has a broad-specificity class I aspartate aminotransferase (48) as well as a homolog of the class I aspartate aminotransferase found in the crenarchaeon Sulfolobus solfataricus (6). The diverse substrate specificities of the class IV aminotransferases and the apparent convergent evolution of aspartate aminotransferase activity in at least three different classes of aminotransferases demonstrate the difficulty in predicting enzyme function for highly similar members of a protein family (51).

The evolution of the methanogenic class V aminotransferase family parallels the evolution of the tRNA-dependent cysteine biosynthetic pathway. Most methanogenic euryarchaea that carry this AspAT also have homologs of the O-phosphoseryl-tRNA synthetase and the Sep-tRNA:Cys-tRNA synthase (38). Therefore, these enzymes for phosphoserine and tRNA-dependent cysteine biosynthesis were encoded in the genome of the euryarchaeal methanogen ancestor. The Methanosarcinales acquired canonical cysteine biosynthesis enzymes (phosphoserine transaminase, serine acetyltransferase, O-acetylserine sulfhydrolase, and cysteinyl-tRNA synthetase) by horizontal gene transfer, making the tRNA-dependent pathway redundant (3, 21, 26). Yet these organisms have retained the class V AspAT, suggesting that this multifunctional enzyme plays several roles in cell metabolism.


   ACKNOWLEDGMENTS
 
We thank Kelly Sheppard for experimental help.

This work was supported in part by grant DE-FG02-98ER20311 from the Energy Biosciences Program, U.S. Department of Energy (to D.S.), and by grant F-1576 from the Welch Foundation (to D.G.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, TX 78712. Phone: (512) 471-4491. Fax: (512) 471-8696. E-mail: degraham{at}mail.utexas.edu. Back

{triangledown} Published ahead of print on 27 October 2006. Back


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Journal of Bacteriology, January 2007, p. 575-582, Vol. 189, No. 2
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