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.
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi, 464-8601, Japan.
Tomokazu
T
Ito
Hirotaka
H
Murase
Motoki
M
Maekawa
Masaru
M
Goto
Shuhei
S
Hayashi
Hajime
H
Saito
Masatoshi
M
Maki
Hisashi
H
Hemmi
Tohru
T
Yoshimura
D002118:Calcium / Q000737:chemistry*; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000737:chemistry / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D009154:Mutation; D011732:Pyridoxal Phosphate / Q000737:chemistry; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000737:chemistry / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D012964:Sodium / Q000737:chemistry*; D013237:Stereoisomerism; D014867:Water / Q000737:chemistry; D000099300:Serine Racemase
D002118:Calcium; D002413:Cations, Divalent; D002414:Cations, Monovalent; D008274:Magnesium; D011732:Pyridoxal Phosphate; D019998:Racemases and Epimerases; D011994:Recombinant Proteins; D012694:Serine; D012964:Sodium; D014867:Water; D000099300:Serine Racemase