Metal ion dependency of serine racemase from Dictyostelium discoideum

Abstract

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.

Distribution of eukaryotic serine racemases in the bacterial domain and characterization of a representative protein in Roseobacter litoralis Och 149

“…3). In addition, Na + and K + (20 mM) stimulated RiSR racemization activity, but did not affect dehydration activity, similar to the effects of these cations on the mouse SR (Ito et al, 2012). Taken together, our data suggested that the effects of metal ions and chemicals on RiSR activities and the specific conserved amino acid residues involved in MgATP binding were similar to those of eukaryotic SRs, particularly mammalian SRs.…”

“…In this analysis, six amino acid sequences of SRs with known functions were selected as query sequences: one bacterial SR (UniProt accession no. Q9|3P3, Enterococcus gallinarum; Arias et al, 1999), one BSR (Q88GF8, P. putida; Asano & Endo, 1988) and four eukaryotic SRs (Q9GZT4, Homo sapiens; Q2PGG3, Arabidopsis thaliana; Q54HH2, Dictyostelium discoideum; and O59791, Schizosaccharomyces pombe; De Miranda et al, 2000;Hoffman et al, 2009;Fujitani et al, 2006;Ito et al, 2012;Goto et al, 2009). In addition, we used two sequences of THADH (A4F2N8, Pseudomonas sp.…”

“…D-serine is synthesized from L-serine by serine racemases (SRs), which belong to a group of pyridoxal 59-phosphate (PLP)-dependent enzymes (Grishin et al, 1995;Yoshimura & Esak, 2003;Yoshimura & Goto, 2008). Currently, many genes encoding eukaryotic SRs in humans (De Miranda et al, 2000;Hoffman et al, 2009), mice (Foltyn et al, 2005;Strísovský et al, 2005), plants (Fujitani et al, 2006(Fujitani et al, , 2007Gogami et al, 2010), slime moulds (Ito et al, 2012) and fission yeasts (Goto et al, 2009) have been cloned, and the enzymatic properties of these proteins have been characterized.…”

Conserved Pyridoxal Protein That Regulates Ile and Val Metabolism

“…Amino acid mixture standard solution (Type H; Wako, Japan) was used for identification and quantification of amino acids. The enantioselective amino acid analysis was performed as described previously (8,9).…”

Crystal structure of a pyridoxal 5′-phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii

“…In addition, it has been shown that d-aspartate in the bivalve mollusc Scapharca broughtonii is produced by a PLP-dependent AspR (SbAspR) similar to mammalian SR (Shibata et al, 2003). The amino-acid sequence of SbAspR shows approximately 42.7% identity to that of rat SR and 36.6% identity to that of yeast SR. SR is activated by ATP (de Miranda et al, 2002;Neidle & Dunlop, 2002;Ito et al, 2012), whereas the activity of SbAspR is increased and decreased by AMP and ATP, respectively (Shibata et al, 2003). X-ray crystallographic studies have revealed the ATP-binding sites in human, rat and yeast SRs (Goto et al, 2009;Smith et al, 2010).…”