Mechanism of intrinsic resistance to vancomycin in Clostridium innocuum NCIB 10674

We have studied the basis for intrinsic resistance to low levels of vancomycin in Clostridium innocuum NCIB 10674 (MIC = 8 microg/ml). Analysis by high-pressure liquid chromatography (HPLC) and mass spectrometry of peptidoglycan nucleotide precursors pools revealed the presence of two types of UDP-MurNac-pentapeptide precursors constitutively produced, an UDP-MurNAc-pentapeptide with a serine at the C terminus which represented 93% of the pool and an UDP-MurNAc-pentapeptide with an alanine at the C terminus which represented the rest of the pool. C. innocuum cell wall muropeptides containing pentapeptide[Ser], either dialanine substituted on the epsilon amino group of lysine or not, were identified and represented about 10% of the monomers while only 1% of pentapeptide[D-Ala] monomers were found. The sequence of a 2,465-bp chromosomal fragment from C. innocuum was determined and revealed the presence of ddl(c. innocuum) and C. innocuum racemase genes putatively encoding homologues of D-Ala:D-X ligases and amino acid racemases, respectively. Analysis of the pool of precursors of Enterococcus faecalis JH2-2, containing cloned ddl(c. innocuum) and C. innocuum racemase genes showed in addition to the UDP-MurNAc-pentapeptide[D-Ala], the presence of an UDP-MurNAc-pentapeptide[D-Ser] precursor. However, the expression of low-level resistance to vancomycin was observed only when both genes were cloned in E. faecalis JH2-2 together with the vanXYc gene from Enterococcus gallinarum BM4174 which encodes a d,d-peptidase which eliminates preferentially the high affinity vancomycin UDP-MurNAc-pentapeptide [D-Ala] precursors produced by the host. We conclude that resistance to vancomycin in C. innocuum NCIB 10674 was related to the presence of the two chromosomal ddl(c. innocuum) and C. innocuum racemase genes allowing the synthesis of a peptidoglycan precursor terminating in serine with low affinity for vancomycin.     

Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine:D-alanine ligase-related protein necessary for vancomycin resistance.

The amplification product obtained with DNA from vancomycin-resistant (VmR) Enterococcus gallinarum BM4174 and a pair of degenerate oligodeoxyribonucleotides that correspond to conserved amino acid (aa) motifs in Escherichia coli D-alanine (D-Ala):D-Ala ligases and in En. faecium VmR protein (VanA) was used as a probe to clone the vanC gene of that strain. The vanC product, with a calculated Mr of 37,504, exhibits 29 to 38% aa identity with VanA and E. coli ligases. Insertional inactivation of vanC led to Vm sensitivity of BM4174 suggesting that the gene may encode a D-Ala:D-Ala ligase of altered specificity.     

Vancomycin resistance is associated with serine-containing peptidoglycan in Enterococcus gallinarum

In Enterococcus gallinarum SC1, a low-level vancomycin-resistant strain, only monomeric muropentapeptides with a C-terminal D-alanine were detected after growth without vancomycin. In contrast, in SC1 induced by vancomycin, as well as in AIB39, a constitutive vancomycin-resistant strain, monomeric and dimeric muropentapeptides with a C-terminal D-serine were detected.     

Serine racemase modulates intracellular D-serine levels through an alpha,beta-elimination activity

Mammalian brain contains high levels of d-serine, an endogenous co-agonist of N-methyl D-aspartate type of glutamate receptors. D-Serine is synthesized by serine racemase, a brain enriched enzyme converting L- to D-serine. Degradation of D-serine is achieved by D-amino acid oxidase, but this enzyme is not present in forebrain areas that are highly enriched in D-serine. We now report that serine racemase catalyzes the degradation of cellular D-serine itself, through the alpha,beta-elimination of water. The enzyme also catalyzes water alpha,beta-elimination with L-serine and L-threonine. alpha,beta-Elimination with these substrates is observed both in vitro and in vivo. To investigate further the role of alpha,beta-elimination in regulating cellular D-serine, we generated a serine racemase mutant displaying selective impairment of alpha,beta-elimination activity (Q155D). Levels of D-serine synthesized by the Q155D mutant are several-fold higher than the wild-type both in vitro and in vivo. This suggests that the alpha,beta-elimination reaction limits the achievable D-serine concentration in vivo. Additional mutants in vicinal residues (H152S, P153S, and N154F) similarly altered the partition between the alpha,beta-elimination and racemization reactions. alpha,beta-Elimination also competes with the reverse serine racemase reaction in vivo. Although the formation of L- from D-serine is readily detected in Q155D mutant-expressing cells incubated with physiological D-serine concentrations, reversal with wild-type serine racemase-expressing cells required much higher D-serine concentration. We propose that alpha,beta-elimination provides a novel mechanism for regulating intracellular D-serine levels, especially in brain areas that do not possess D-amino acid oxidase activity. Extracellular D-serine is more stable toward alpha,beta-elimination, likely due to physical separation from serine racemase and its elimination activity.     

Human serine racemase: moleular cloning, genomic organization and functional analysis

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.     

Modulation of D-serine levels via ubiquitin-dependent proteasomal degradation of serine racemase

Mammalian serine racemase is a brain-enriched enzyme that converts L- into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain d-serine levels and NMDA receptor activity.     

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.     

D-amino acids in the brain: the biochemistry of brain serine racemase

It has been recently established that in various brain regions D-serine, the product of serine racemase, occupies the so-called 'glycine site' within N-methyl D-aspartate receptors. Mammalian brain serine racemase is a pyridoxal-5' phosphate-containing enzyme that catalyzes the racemization of L-serine to D-serine. It has also been shown to catalyze the alpha,beta-elimination of water from L-serine or D-serine to form pyruvate and ammonia. Serine racemase is included within the group of type II-fold pyridoxal-5' phosphate enzymes, together with many other racemases and dehydratases. Serine racemase was first purified from rat brain homogenates and later recombinantly expressed in mammalian and insect cells as well as in Escherichia coli. It has been shown that serine racemase is activated by divalent cations like calcium, magnesium and manganese, as well as by nucleotides like ATP, ADP or GTP. In turn, serine racemase is also strongly inhibited by reagents that react with free sulfhydryl groups such as glutathione. Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein interacting with C kinase 1 and Golga3 to bind to serine racemase, having different effects on its catalytic activity or stability. In addition, it has also been proposed that serine racemase is regulated by phosphorylation. Thus, d-serine production in the brain is tightly regulated by various factors pointing at its physiologic importance. In this minireview, we will focus on the regulation of brain serine racemase and d-serine synthesis by the factors mentioned above.     

D-Serine as a putative glial neurotransmitter

Abundant recent evidence favors a neurotransmitter/neuromodulator role for D-serine. D-serine is synthesized from L-serine by serine racemase in astrocytic glia that ensheath synapses, especially in regions of the brain that are enriched in NMDA-glutamate receptors. D-serine is more potent than glycine at activating the 'glycine' site of these receptors. Moreover, selective degradation of D-serine but not glycine by D-amino acid oxidase markedly reduces NMDA neurotransmission. D-serine appears to be released physiologically in response to activation by glutamate of AMPA-glutamate receptors on D-serine-containing glia. This causes glutamate-receptor-interacting protein, which binds serine racemase, to stimulate enzyme activity and D-serine release. Thus, glutamate triggers the release of D-serine so that the two amino acids can act together on postsynaptic NMDA receptors. D-serine also plays a role in neural development, being released from Bergmann glia to chemokinetically enhance the migration of granule cell cerebellar neurons from the external to the internal granular layer.     

Neuron-derived D-serine release provides a novel means to activate N-methyl-D-aspartate receptors

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.     

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.     

Neurobiology through the looking-glass: D-serine as a new glial-derived transmitter

D-Amino acids have been known to be present in bacteria for more than 50 years, but only recently they were identified in mammals. The occurrence of D-amino acids in mammals challenge classic concepts in biology in which only L-amino acids would be present or thought to play important roles. Recent discoveries uncovered a role of endogenous D-serine as a putative glial-derived transmitter that regulates glutamatergic neurotransmission in mammalian brain. Free D-serine levels in the brain are about one third of L-serine values and its extracellular concentration is higher than many common L-amino acids. D-Serine occurs in protoplasmic astrocytes, a class of glial cells that ensheath the synapses and modulate neuronal activity. Biochemical and electrophysiological studies suggest that endogenous D-serine is a physiological modulator at the co-agonist site of NMDA-type of glutamate receptors. We previously showed that D-serine is synthesized by a glial serine racemase, a novel enzyme converting L- to D-serine in mammalian brain. The enzyme requires pyridoxal 5'-phosphate and it was the first racemase to be cloned from eucaryotes. Inhibitors of serine racemase have therapeutic implications for pathological processes in which over-stimulation of NMDA receptors takes place, such as stroke and neurodegenerative diseases. Here, we review the role of endogenous D-serine in modulating NMDA neurotransmission, its biosynthetic apparatus and the potential usefulness of serine racemase inhibitors as a novel neuroprotective strategy to decrease glutamate/NMDA excitotoxicity.     

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.     

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases

Pyridoxal-5′-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.

     

Gene cloning,purification, and characterization of 2,3-diaminopropionate ammonia-lyase from Escherichia coli

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes alpha,beta-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5'-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the alpha,beta-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D-and L-beta-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.     

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.     

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.