Insights into the structural properties of D-serine dehydratase from Saccharomyces cerevisiae: an FT-IR spectroscopic and in silico approach

d-serine dehydratase (Dsd) from baker’s yeast is a recently discovered enzyme catalyzing the oxidation of d-serine to pyruvate and ammonia. The reaction depends on the cofactors pyridoxal-5′-phosphate (PLP) and Zn²⁺, featuring a very high selectivity towards the d-enantiomer of the amino acid serine. In humans, altered levels of d-serine in the cerebrospinal fluid (CSF) and blood correlate with the onset and evolution of a number of neurodegenerative diseases. Up to date very little is known on the structure of Dsd. Hence, we have investigated the structure of this enzyme by means of Fourier Transform infrared (FT-IR) spectroscopy and used the structural data derived thereof to validate a homology model of Dsd. In this model, Dsd adopts a fold that is characteristic of type III pyridoxal-dependent enzymes. This consists of an α/β (TIM) barrel and a β-sandwich domain at the N- and C-termini, respectively. Analysis of the Amide I and Amide III infrared bands revealed that the amounts of α (24%), β (29%) and unordered structures (47%) correlate well with those derived from the model (25%, 29% and 46% respectively), suggesting reliability of the latter. In addition, the model of Dsd was further refined by recreating the PLP- and zinc-restored active site based on a PLP- and zinc-dependent bacterial amino acid racemase recently crystallized, allowing us to identify the potential cofactor and metal binding residues of Dsd.     

Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The Vmax/Km values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana.     

Crystal structure of D-serine dehydratase from Escherichia coli

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97?-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55? resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.     

Rapid determination of free D-serine with chicken D-serine dehydratase

We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free D-serine in tissue samples. This method uses chicken D-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5'-phosphate (PLP), a cofactor of D-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn(2+) or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of L-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2-30 μM (200 pmol-3 nmol) of D-serine. The entire assay requires only 60 min. With this method, we determined the concentration of D-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of D-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g(-1) wet tissue in the midgut, 434 nmol g(-1) in the ovary, and 353 nmol g(-1) in the testis.     

Crystal structure of D-serine dehydratase from Escherichia coli

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5′-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97 Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55 Å resolution. The structure of DSD reveals a larger pyridoxal 5′-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.     

Occurrence of D-serine in rice and characterization of rice serine racemase

Germinated, unpolished rice was found to contain a substantial amount of D-serine, with the ratio of the D-enantiomer to the L-enantiomer being higher for serine than for other amino acids. The relative amount of D-serine (D/(D + L)%) reached approximately 10% six days after germination. A putative serine racemase gene (serr, clone No. 001-110-B03) was found in chromosome 4 of the genomic DNA of Oryza sativa L. ssp. Japonica cv. Nipponbare. This was expressed as serr in Escherichia coli and its gene product (SerR) was purified to apparent homogeneity. SerR is a homodimer with a subunit molecular mass of 34.5 kDa, and is highly specific for serine. In addition to a serine racemase reaction, SerR catalyzes D- and L-serine dehydratase reactions, for which the specific activities were determined to be 2.73 and 1.42 nkatal/mg, respectively. The optimum temperature and pH were respectively determined for the racemase reaction (35 °C and pH 9.0) and for the dehydratase reaction (35 °C and pH 9.5). SerR was inhibited by PLP-enzyme inhibitors. ATP decreased the serine racemase activity of SerR but increased the serine dehydratase activity. Kinetic analysis showed that Mg²⁺ increases the catalytic efficiency of the serine racemase activity of SerR and decreases that of the serine dehydratase activity. Fluorescence-quenching analysis of the tryptophan residues in SerR indicated that the structure of SerR is distorted by the addition of Mg²⁺, and this structural change probably regulates the two enzymatic activities.     

Ionization state of pyridoxal 5'-phosphate in D-serine dehydratase, dialkylglycine decarboxylase and tyrosine phenol-lyase and the influence of monovalent cations as inferred by 31P NMR spectroscopy

The 31P NMR spectroscopy of three pyridoxal 5'-phosphate-dependent enzymes, monomeric D-serine dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied. 31P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by approximately 3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The 31P NMR signal of the phosphate group of pyridoxal 5'-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. For D-serine dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from 31P NMR data.     

Metagenome resource for D-serine utilization in a DsdA-disrupted Escherichia coli

To find alternative genetic resources for D-serine dehydratase (E.C. 4.3.1.18, dsdA) mediating the deamination of D-serine into pyruvate, metagenomic libraries were screened. The chromosomal dsdA gene of a wild-type Escherichia coli W3110 strain was disrupted by inserting the tetracycline resistance gene (tet), using double-crossover, for use as a screening host. The W3110 dsdA::tet strain was not able to grow in a medium containing D-serine as a sole carbon source, whereas wild-type W3110 and the complement W3110 dsdA::tet strain containing a dsdA-expression plasmid were able to grow. After introducing metagenome libraries into the screening host, a strain containing a 40-kb DNA fragment obtained from the metagenomic souce derived from a compost was selected based on its capability to grow on the agar plate containing D-serine as a sole carbon source. For identification of the genetic resource responsible for the D-serine degrading capability, transposon- micron was randomly inserted into the 40-kb metagenome. Two strains that had lost their D-serine degrading ability were negatively selected, and the two 6-kb contigs responsible for the D-serine degrading capability were sequenced and deposited (GenBank code: HQ829474.1 and HQ829475.1). Therefore, new alternative genetic resources for D-serine dehydratase was found from the metagenomic resource, and the corresponding ORFs are discussed.     

Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase

We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase. These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites. The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid). The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides. C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively. The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity. The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes). C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase. Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A. These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site.     

A novel zinc-dependent D-serine dehydratase from Saccharomyces cerevisiae

YGL196W of Saccharomyces cerevisiae encodes a putative protein that is unidentified but is predicted to have a motif similar to that of the N-terminal domain of the bacterial alanine racemase. In the present study we found that YGL196W encodes a novel D-serine dehydratase, which belongs to a different protein family from that of the known bacterial enzyme. The yeast D-serine dehydratase purified from recombinant Escherichia coli cells depends on pyridoxal 5'-phosphate and zinc, and catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. D-Threonine and beta-Cl-D-alanine also serve as substrates with catalytic efficiencies which are approx. 3 and 2% of D-serine respectively. L-Serine, L-threonine and beta-Cl-L-alanine are inert as substrates. Atomic absorption analysis revealed that the enzyme contains one zinc atom per enzyme monomer. The enzyme activities toward D-serine and D-threonine were decreased by EDTA treatment and recovered by the addition of Zn2+. Little recovery was observed with Mg2+, Mn2+, Ca2+, Ni2+, Cu2+, K+ or Na+. In contrast, the activity towards beta-Cl-D-alanine was retained after EDTA treatment. These results suggest that zinc is involved in the elimination of the hydroxy group of D-serine and D-threonine. D-Serine dehydratase of S. cerevisiae is probably the first example of a eukaryotic D-serine dehydratase and that of a specifically zinc-dependent pyridoxal enzyme as well.     

D-serine-dehydratase from Saccaromyces cerevisiae: a pyridoxal 5'- phosphate-dependent enzyme for advanced biotech applications

The Saccaromices cerevisiae D-serine dehydratase is a pyridoxal 5'-phosphate dependent enzyme that requires zinc for its function. It catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. In this work, a new methodology for monitoring D-serine is presented. Our results show that this enzyme could be successfully used as a biological probe for detection of D-serine via fluorescence spectroscopy.     

Contribution of a conserved arginine near the active site of Escherichia coli D-serine dehydratase to cofactor affinity and catalytic activity

We have employed site-directed mutagenesis to investigate the contribution of a conserved arginyl residue to the catalytic activity and cofactor affinity of D-serine dehydratase, a model pyridoxal 5'-phosphate (vitamin B6) enzyme. Replacement of R-120 in the active site peptide of D-serine dehydratase by L decreased the affinity of the enzyme for pyridoxal 5'-phosphate by 20-fold and reduced turnover by 5-8-fold. kappa cat displayed modified substrate alpha-deuterium isotope effects and altered dependence on both temperature and pH. Analysis of the pH rate profiles of DSD and the R-120----L variant indicated that R-120 interacts electrostatically with catalytically essential ionizable groups at the active site of wild type D-serine dehydratase. The decrease in cofactor affinity observed for DSD(R120L) was not accompanied by significant perturbations in the UV, CD, or 31P NMR spectrum of the holoenzyme, suggesting that the contribution of R-120 to pyridoxal phosphate affinity may be indirect or else involve an interaction with a cofactor functional group other than the 5'-phosphoryl moiety. The properties of two other site-directed variants of D-serine dehydratase indicated that the pyridoxal 5'-phosphate:K-118 Schiff base was indifferent to a small change in the shape of the side chain at position 117 (I-117----L), whereas replacement of K-118 by H resulted in undetectable levels of enzyme. A poor ability to bind cofactor may have rendered DSD(K118H) susceptible to intracellular proteolysis.     

The glycine-rich region of Escherichia coli D-serine dehydratase. Altered interactions with pyridoxal 5'-phosphate produced by substitution of aspartic acid for glycine

Replacement of glycine by aspartic acid at either of two sites in a conserved, glycine-rich region inactivates the pyridoxal 5'-phosphate-dependent enzyme D-serine dehydratase (DSD) from Escherichia coli. To investigate why aspartic acid at position 279 or 281 causes a loss of activity, we measured the affinity of the G----D variants for pyridoxal 5'-phosphate and a cofactor:substrate analog complex and compared the UV, CD, and fluorescence properties of wild-type D-serine dehydratase and the inactive variants. The two G----D variants DSD(G279D) and DSD (G281D) displayed marked differences from wild-type D-serine dehydratase and from each other with respect to their affinity for pyridoxal 5'-phosphate and for a pyridoxal 5'-phosphate:glycine Schiff base. Compared to the wild-type enzyme, the cofactor affinity of DSD(G279D) and DSD(G281D) was decreased 225- and 50-fold, respectively, and the ability to retain a cofactor:glycine complex was decreased 765- and 1970-fold. The spectral properties of the inactive variants suggest that they form a Schiff base linkage with pyridoxal 5'-phosphate but do not hold the cofactor in a catalytically competent orientation. Moreover, the amount of cofactor aldamine in equilibrium with cofactor Schiff base is increased in DSD(G279D) and DSD(G281D) relative to that in wild-type DSD. Collectively, our findings indicate that introduction of a carboxymethyl side chain at G-279 or G-281 directly or indirectly disrupts catalytically essential protein-cofactor and protein-substrate interactions and thereby prevents processing of the enzyme bound cofactor:substrate complex. The conserved glycine-rich region is thus either an integral part of the D-serine dehydratase active site or conformationally linked to it.     

Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor

HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.     

Mechanism of action of D-serine dehydratase. Identification of a transient intermediate.

Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2 degrees C show that during the steady-state course of D-serine conversion the absorption maximum of the Schiff base of the cofactor pyridoxal 5'-phosphate (pyridoxal-P) is shifted from 415 to 442 nm. Furthermore, the progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320 to 500 nm. A point by point construction of successive spectra from these stopped-flow traces at various time intervals after the start of reaction resulted in a series of consecutive spectra exhibiting two isobestic points at 353 and 419 nm. The half-time of the absorbance changes occurring at 330 and 455 nm was found to be 6.5 ms, suggesting the observation of a single, enzyme-bound intermediate. The spectral data with substrate and inhibitors provide evidence that the intermediate is the Schiff base of alpha-aminoacrylate and pyridoxal-P. The proposed assignment is strongly supported by experiments of apodehydratase with transient-state analogues which exhibit a similar absorbance shift on binding to apoenzyme. Moreover, these results suggest that the phosphate group of the substrate--pyridoxal-P complex serves as the main anchoring point during catalysis. A reaction mechanism of the D-serine dehydratase is presented.     

Effect of potassium ion on the phosphorus-31 nuclear magnetic resonance spectrum of the pyridoxal 5'-phosphate cofactor of Escherichia coli D-serine dehydratase

31P NMR studies were undertaken to determine how potassium ion increases the cofactor affinity of Escherichia coli D-serine dehydratase, a model pyridoxal 5'-phosphate requiring enzyme that converts the growth inhibitor D-serine to pyruvate and ammonia. Potassium ion was shown to promote the appearance of a second upfield shifted cofactor 31P resonance at 4.0 ppm (pH 7.8, 25 degrees C), that increased in area at the expense of the resonance at 4.4 ppm observed in the absence of K+. Na+ antagonized the K+ promoted appearance of the second resonance. These observations suggest that K+ and Na+ stabilize conformational states that differ with respect to O-P-O bond angle, conformation, and/or hydrogen bonding of the phosphate group. An analysis of the dependence of the relative intensities of the two resonances on the K+ concentration yielded a value of ca. 10 mM for the equilibrium constant for dissociation of K+ from D-serine dehydratase. The chemical shift difference between the two resonances indicated that the K+-stabilized and Na+-stabilized forms of the enzyme interconvert at a frequency less than 16 s-1 at pH 7.8, 25 degrees C.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.     

Protein kinase C activity regulates D-serine availability in the brain

D-serine is a co-agonist of NMDA receptor (NMDAR) and plays important roles in synaptic plasticity mechanisms. Serine racemase (SR) is a brain-enriched enzyme that converts L-serine to D-serine. SR interacts with the protein interacting with C-kinase 1 (PICK1), which is known to direct protein kinase C (PKC) to its targets in cells. Here, we investigated whether PKC activity regulates SR activity and D-serine availability in the brain. In vitro, PKC phosphorylated SR and decreased its activity. PKC activation increased SR phosphorylation in serine residues and reduced D-serine levels in astrocyte and neuronal cultures. Conversely, PKC inhibition decreased basal SR phosphorylation and increased cellular D-serine levels. In vivo modulation of PKC activity regulated both SR phosphorylation and D-serine levels in rat frontal cortex. Finally, rats that completed an object recognition task showed decreased SR phosphorylation and increased D-serine/total serine ratios, which was markedly correlated with decreased PKC activity in both cortex and hippocampus. Results indicate that PKC phosphorylates SR in serine residues and regulates D-serine availability in the brain. This interaction may be relevant for the regulation of physiological and pathological mechanisms linked to NMDAR function.     

D-amino acids in the brain: structure and function of pyridoxal phosphate-dependent amino acid racemases

D-serine serves as a co-agonist of the N-methyl D-aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer's disease and amyotrophic lateral sclerosis. D-Serine is synthesized by a pyridoxal 5'-phosphate (PLP)-dependent serine racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP-dependent amino acid racemase on the basis of its 3D structure. We compared the eukaryotic serine racemase with bacterial alanine racemase, the best-studied enzyme among the PLP-dependent amino acid racemases, and thus suggested a putative reaction mechanism for mammalian D-serine synthesis.     

Discovery and characterization of d-phenylserine deaminase from Arthrobacter sp. TKS1

We discovered a D-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from D-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55°C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50°C. The enzyme acted on the D-forms of β-hydroxy-α-amino acids, such as D-threo-phenylserine (K(m), 19 mM), D-serine (K(m), 5.8 mM), and D-threonine (K(m), 102 mM). As L-threonine, D-allo-threonine, L-allo-threonine, and DL-erythro-phenylserine were inert, the enzyme could distinguish D-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO(4), CuSO(4), BaCl(2), and CoCl(2) and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and DL-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.