D-serine Dehydratase Research Articles

Bacteria use a catabolic patchwork pathway of apparently recent origin for degradation of the synthetic buffer compound TRIS.

The synthetic buffer compound TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) is used in countless applications, and no detailed information on its degradation has been published so far. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, a Pseudomonas strain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase (mSHMT) and a d-serine dehydratase that plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Conjugational plasmid transfer into Pseudomonas putida KT2440 enabled this strain to grow with TRIS and with 2-hydromethylserine, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from the Pseudomonas and Shinella genera carrying highly similar TRIS degradation gene clusters. Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways, and database searches suggest that the TRIS degradation pathway is now globally distributed. Overall, our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales.

Novel tetrahydrofolate-dependent d-serine dehydratase activity of serine hydroxymethyltransferases.

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas thatof E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.

Commentary on "UrinaryL-erythro-β-hydroxyasparagine - a novel serine racemase inhibitor and substrate of the Zn2+-dependentD-serine dehydratase".

The analysis of the urine contents can be informative of physiological homeostasis, and it has been speculated that the levels of urinary D-serine (D-ser) could inform about neurological and renal disorders. By analysing the levels of urinary D-ser using a D-ser dehydratase (DSD) enzyme, Ito et al. have described abundant levels of L-β-EHAsn, a non-proteogenic amino acid which is also a newly described substrate for DSD. The data presented supports the endogenous production L-β-EHAsn, with its concentration significantly correlating with the concentration of creatinine in urine. Taken together, these results could raise speculations that L-β-EHAsn might have unexplored important biological roles. It has been demonstrated that L-β-EHAsn also inhibits serine racemase with Ki values (40 μM) similar to its concentration in urine (50 μM). Given that serine racemase is the enzyme involved in the synthesis of D-ser, and L-β-EHAsn is also a substrate for DSD, further investigations could verify if this amino acid would be involved in the metabolic regulation of pathways involving D-ser.

Urinary l-erythro-β-hydroxyasparagine-a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase.

In the present study, we identified l-erythro-β-hydroxyasparagine (l-β-EHAsn) found abundantly in human urine, as a novel substrate of Zn2+-dependent d-serine dehydratase (DSD). l-β-EHAsn is an atypical amino acid present in large amounts in urine but rarely detected in serum or most organs/tissues examined. Quantitative analyses of urinary l-β-EHAsn in young healthy volunteers revealed significant correlation between urinary l-β-EHAsn concentration and creatinine level. Further, for in-depth analyses of l-β-EHAsn, we developed a simple three-step synthetic method using trans-epoxysuccinic acid as the starting substance. In addition, our research revealed a strong inhibitory effect of l-β-EHAsn on mammalian serine racemase, responsible for producing d-serine, a co-agonist of the N-methyl-d-aspartate (NMDA) receptor involved in glutamatergic neurotransmission.

Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging

Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can then produce lactate from pyruvate. Here, we developed single fluorescent protein (FP)-based lactate and pyruvate indicators with low EC50 for trace detection of metabolic molecules and live cell imaging and named them “Green Lindoblum” and “Green Pegassos,” respectively. Green Lindoblum (EC50 of 30 µM for lactate) and Green Pegassos (EC50 of 70 µM for pyruvate) produced a 5.2- and 3.3-fold change in fluorescence intensity in response to lactate and pyruvate, respectively. Green Lindoblum measured lactate levels in mouse plasma, and Green Pegassos in combination with D-serine dehydratase successfully estimated D-serine levels released from mouse primary cultured neurons and astrocytes by measuring pyruvate level. Furthermore, live cell imaging analysis revealed their utility for dual-colour imaging, and the interplay between lactate, pyruvate, and Ca2+ in human induced pluripotent stem cell-derived cardiomyocytes. Therefore, Green Lindoblum and Green Pegassos will be useful tools that detect specific molecules in clinical use and monitor the interplay of metabolites and other related molecules in diverse cell types.

D-Serine Degradation by Proteus mirabilis Contributes to Fitness during Single-Species and Polymicrobial Catheter-Associated Urinary Tract Infection.

Proteus mirabilis is a common cause of catheter-associated urinary tract infection (CAUTI) and secondary bacteremia, which are frequently polymicrobial. We previously utilized transposon insertion-site sequencing (Tn-Seq) to identify novel fitness factors for colonization of the catheterized urinary tract during single-species and polymicrobial infection, revealing numerous metabolic pathways that may contribute to P. mirabilis fitness regardless of the presence of other cocolonizing organisms. One such "core" fitness factor was d-serine utilization. In this study, we generated isogenic mutants in d-serine dehydratase (dsdA), d-serine permease (dsdX), and the divergently transcribed activator of the operon (dsdC) to characterize d-serine utilization in P. mirabilis and explore the contribution of this pathway to fitness during single-species and polymicrobial infection. P. mirabilis was capable of utilizing either d- or l-serine as a sole carbon or nitrogen source, and dsdA, dsdX, and dsdC were each specifically required for d-serine degradation. This capability was highly conserved among P. mirabilis isolates, although not universal among uropathogens: Escherichia coli and Morganella morganii utilized d-serine, while Providencia stuartii and Enterococcus faecalis did not. d-Serine utilization did not contribute to P. mirabilis growth in urine ex vivo during a 6-h time course but significantly contributed to fitness during single-species and polymicrobial CAUTI during a 96-h time course, regardless of d-serine utilization by the coinfecting isolate. d-Serine utilization also contributed to secondary bacteremia during CAUTI as well as survival in a direct bacteremia model. Thus, we propose d-serine utilization as a core fitness factor in P. mirabilis and a possible target for disruption of infection.IMPORTANCE Urinary tract infections are among the most common health care-associated infections worldwide, the majority of which involve a urinary catheter (CAUTI). Our recent investigation of CAUTIs in nursing home residents identified Proteus mirabilis, Enterococcus species, and Escherichia coli as the three most common organisms. These infections are also often polymicrobial, and we identified Morganella morganii, Enterococcus species, and Providencia stuartii as being more prevalent during polymicrobial CAUTI than single-species infection. Our research therefore focuses on identifying "core" fitness factors that are highly conserved in P. mirabilis and that contribute to infection regardless of the presence of these other organisms. In this study, we determined that the ability to degrade d-serine, the most abundant d-amino acid in urine and serum, strongly contributes to P. mirabilis fitness within the urinary tract, even when competing for nutrients with another organism. d-Serine uptake and degradation therefore represent potential targets for disruption of P. mirabilis infections.

Understanding phospholipid function: Why are there so many lipids?

In the 1970s, phospholipids were still considered mere building blocks of the membrane lipid bilayer, but the subsequent realization that phospholipids could also serve as second messengers brought new interest to the field. My own passion for the unique amphipathic properties of lipids led me to seek other, non-signaling functions for phospholipids, particularly in their interactions with membrane proteins. This seemed to be the last frontier in protein chemistry and enzymology to be conquered. I was fortunate to find my way to Eugene Kennedy's laboratory, where both membrane proteins and phospholipids were the foci of study, thus providing a jumping-off point for advancing our fundamental understanding of lipid synthesis, membrane protein biosynthesis, phospholipid and membrane protein trafficking, and the cellular roles of phospholipids. After purifying and characterizing enzymes of phospholipid biosynthesis in Escherichia coli and cloning of several of the genes encoding these enzymes in E. coli and Saccharomyces cerevisiae, I was in a position to alter phospholipid composition in a systematic manner during the cell cycle in these microorganisms. My group was able to establish, contrary to common assumption (derived from the fact that membrane proteins retain activity in detergent extracts) that phospholipid environment is a strong determining factor in the function of membrane proteins. We showed that molecular genetic alterations in membrane lipid composition result in many phenotypes, and uncovered direct lipid-protein interactions that govern dynamic structural and functional properties of membrane proteins. Here I present my personal "reflections" on how our understanding of phospholipid functions has evolved.

The Cryptic dsdA Gene Encodes a Functional d-Serine Dehydratase in Pseudomonas aeruginosa PAO1

D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution.

A Shear horizontal surface acoustic wave biosensor for a rapid and specific detection of d-serine

d-serine is an amino acid that has an important role in the regulation of N-methyl-d-aspartate (NMDA) signalling in the central nervous system. In fact, it acts as an endogenous co-agonist molecule for the NMDA receptor activation. Since an abnormal body level of d-amino acids is usually associated to different neurological diseases, it appears evident that it is of high interest to develop a fast and specific biosensor for the determination of d-serine.In this work, we report on the design and development of a surface acoustic wave (SAW) biosensor to detect the level of d-serine. The SAW-biosensor is based on the utilization of an inactivated form of the d-serine dehydratase isolated from Saccharomyces cerevisiae, a biomolecule that is able to specifically bind d-serine. In particular, we produced a PLP-reduced d-serine dehydratase biomolecule that was still able to bind d-serine and not to transform it into the product. The binding event between the “inactivated” d-serine dehydratase and d-serine was monitored by SAW delay-lines.

PEGylated d-serine dehydratase as a d-serine reducing agent

d-Serine is an endogenous coagonist for N-methyl-d-aspartate (NMDA) receptors and is involved in excitatory neurotransmission. Excessive receptor activation causes excitotoxicity, leading to various acute and chronic neurological disorders. Decrease in d-serine content may provide a therapeutic strategy for the treatment of the neurological disorders in which overstimulation of NMDA receptors plays a pathological role. Saccharomyces cerevisiaed-serine dehydratase (Dsd1p), which acts dominantly on d-serine, may be a useful d-serine reducing agent. We conjugated a linear 5-kDa polyethylene glycol (PEG) to Dsd1p (PEG-Dsd1p) and examined the effects of PEG-conjugation on its biochemical and pharmacokinetic properties. PEG-Dsd1p retained activity, specificity, and stability of the enzyme. The PEG modification extended the serum half-life of Dsd1p in mice 6-fold, from 3.8h to 22.4h. PEG-Dsd1p was much less immunogenic compared to the unmodified enzyme. Intraperitoneal administration of PEG-Dsd1p was effective in decreasing the d-serine content in the mouse hippocampus. These findings suggest that PEG-Dsd1p may be a novel tool for lowering d-serine levels in vivo.

Effect of d-serine on spermatogenesis and extracellular signal-regulated protein kinase (ERK) phosphorylation in the testis of the silkworm, Bombyx mori

Although the pupae and larvae of Bombyx mori possess especially large amounts of free d-serine, the physiological role of the amino acid in the silkworm is unknown. We investigated the effect of d-serine on spermatogenesis. A lowered d-serine level throughout larval development caused a delay in spermatogenesis and resulted in reduced numbers of eupyrene sperm. Administration of d-serine transiently increased the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2; hereafter, ERK) by approximately 25% in the testis of day 3 fifth instar larvae. l-Serine had no effect on ERK activation, and other organs did not respond to d-serine. The effect of d-serine on ERK activation was confirmed by administering d-serine dehydratase, an enzyme that specifically degrades d-serine, and the enzyme’s inhibitor, hydroxylamine. ERK phosphorylation in the testis was significantly inhibited by Go6983 and U0126, inhibitors of protein kinase C (PKC) and mitogen-associated protein kinase kinase 1/2 (MEK), respectively, but not by H-89, a protein kinase A (PKA) inhibitor, indicating that ERK was activated in the testis via PKC and MEK but not via PKA. The inhibition of ERK phosphorylation by Go6983 or U0126 was reduced by 20–30% by d-serine. Roughly 30% of c-Raf phosphorylation at an inhibitory site (Ser259) was decreased by the addition of d-serine. These results suggest that d-serine activates ERK in the testis of silkworms through a pathway including c-Raf but not PKC or MEK. Immunohistochemistry confirmed d-serine-induced ERK phosphorylation in the testis and revealed the presence of phospho-ERK in the nuclei of spermatocytes and spermatids.

Reaction mechanism of Zn2+-dependent d-serine dehydratase: role of a conserved tyrosine residue interacting with pyridine ring nitrogen of pyridoxal 5'-phosphate.

d-Serine dehydratase from Saccharomyces cerevisiae (Dsd1p) is a pyridoxal 5'-phosphate (PLP)- and Zn(2+)-dependent enzyme that catalyzes the dehydration of d-serine to yield pyruvate and ammonia. Dsd1p uses the Tyr residue (Y203) to interact with the pyridine nitrogen of PLP, which is a unique feature of PLP enzymes. To investigate the role of Y203 in catalysis, a series of Y203 mutants was constructed and studied. Mutant enzymes possessing a non-polar or a basic residue instead of Y203 (Y203F, A, S and R) exhibited substantial levels of catalytic activity, and among these, the Y203F mutant had the least impact on catalytic activity. The Y203D exhibited a 10(5)-fold decrease in enzyme activity, and unlike wild-type enzyme, the mutant enzyme favoured the Cα reprotonation before hydroxyl group protonation. Our data show that the Y203 does not participate in the protonation of the pyridine nitrogen (N1) of PLP, and Dsd1p uses the cofactor in an N1-unprotonated state. The unprotonated N1 promotes elimination of the leaving group and evades Cα reprotonation before hydroxyl group protonation.

Immunohistochemical localization of d-serine dehydratase in chicken tissues

Chicken d-serine dehydratase (DSD) degrades d-serine to pyruvate and ammonia. The enzyme requires both pyridoxal 5′-phosphate and Zn2+ for its activity. d-Serine is a physiological coagonist that regulates the activity of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate. We have recently found in chickens that d-serine is degraded only by DSD in the brain, whereas it is also degraded to 3-hydroxypyruvate by d-amino acid oxidase (DAO) in the kidney and liver. In mammalian brains, d-serine is degraded only by DAO. It has not been clarified why chickens selectively use DSD for the control of d-serine concentrations in the brain. In the present study, we measured DSD activity in chicken tissues, and examined the cellular localization of DSD using a specific anti-chicken DSD antibody. The highest activity was found in kidney. Skeletal muscles and heart showed no activity. In chicken brain, cerebellum showed about 6-fold-higher activity (1.1±0.3U/g protein) than cerebrum (0.19±0.03U/g protein). At the cellular level DSD was demonstrated in proximal tubule cells of the kidney, in hepatocytes, in Bergmann-glia cells of the cerebellum and in astrocytes. The finding of DSD in glial cells seems to be important because d-serine is involved in NMDAR-dependent brain functions.

Reaction mechanism of yeast D-serine dehydratase

Reaction mechanism of yeast D-serine dehydratase

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.

Metal ion dependency of serine racemase from Dictyostelium discoideum

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2 μM and 2.2 mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.

Role of zinc ion for catalytic activity in d‐serine dehydratase from Saccharomyces cerevisiae

d-Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme catalyzing d-serine dehydration. The enzyme contains 1 mol Zn(2+) in its active site and shows a unique zinc dependence. The Zn(2+) is essential for the d-serine dehydration, but not for the α,β-elimination of β-Cl-d-alanine catalyzed as a side-reaction. The fact that dehydration of d-threonine and d-allo-threonine, also catalyzed by DsdSC, is likewise Zn(2+) dependent indicates that Zn(2+) is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of C(β) . Removal of Zn(2+) results in a less polar active site without changing the gross conformation of DsdSC. (1) H NMR determined the rates of α-hydrogen abstraction and hydroxyl group elimination of d-serine in (2) H(2) O to be 9.7 and 8.5 s(-1) , respectively, while the removal of Zn(2+) abolished both reactions. Mutation of Cys400 or His398 within the Zn(2+) binding sites to Ala endowed DsdSC with similar properties to those of the Zn(2+) -depleted wild-type enzyme: the mutants lost the reactivity toward d-serine and d-threonine but retained that toward β-Cl-d-alanine. (1) H NMR analysis also revealed that both α-hydrogen abstraction and hydroxyl group elimination from d-serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the α-hydrogen abstraction and hydroxyl group elimination in a concerted fashion.

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 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å 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.

Turning pyridoxal-5′-phosphate-dependent enzymes into thermostable binding proteins: d-Serine dehydratase from baker’s yeast as a case study

d-serine dehydratase from Saccharomyces cerevisae is a recently discovered dimeric enzyme catalyzing the β-elimination of d-serine to pyruvate and ammonia. The reaction is highly enantioselective and depends on cofactor pyridoxal-5′-phosphate (PLP) and Zn2+. In our work, the aldimine linkage tethering PLP to recombinant, tagged d-serine dehydratase (Dsd) has been reduced by treatment with NaBH4 so as to yield an inactive form of the holoenzyme (DsdR), which was further treated with a protease in order to remove the amino-terminal purification tag. Fourier Transform infrared (FT-IR) spectroscopic analysis revealed that both the reduced form (DsdR) and the reduced/detagged form (DsdRD) maintain the overall secondary structure of Dsd, but featured a significant increased thermal stability. The observed Tm values for DsdR and for DsdRD shifted to 71.5 °C and 73.3 °C, respectively, resulting in nearly 11 °C and 13 °C higher than the one measured for Dsd. Furthermore, the analysis of the FT-IR spectra acquired in the presence of d-serine and l-serine indicates that, though catalytically inert, DsdRD retains the ability to enantioselectively bind its natural substrate. Sequence analysis of d-serine dehydratase and other PLP-dependent enzymes also highlighted critical residues involved in PLP binding. In virtue of its intrinsic properties, DsdRD represents an ideal candidate for the design of novel platforms based on stable, non-consuming binding proteins aimed at measuring d-serine levels in biological fluids.

Crystal Structure of a Zinc-dependent d-Serine Dehydratase from Chicken Kidney

D-serine is a physiological co-agonist of the N-methyl-D-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, D-amino acid oxidase degrades D-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a D-serine dehydratase degrades D-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated D-threonine aldolases, which are fold-type III pyridoxal 5'-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of D-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on D-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn(2+). None of the reaction products that would be expected from side reactions of the PLP-D-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the D-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His(347) and Cys(349) is located near the PLP-Lys(45) Schiff base. A theoretical model of the enzyme-D-serine complex suggested that the hydroxyl group of D-serine directly coordinates the zinc ion, and that the ε-NH(2) group of Lys(45) is a short distance from the substrate Cα atom. The α-proton abstraction from D-serine by Lys(45) and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.