Department of Biochemistry, Osaka Medical College* Department of Chemistry, Osaka City University, Japan** Institute of Chemical Research, Kyoto University, Japan*** Department of Applied Molecular Bioscience, Nagoya University, Japan****
○Masaru Goto* Ikuko Miyahara** Takae Yamauchi*** Tohru Yoshimura**** Nobuyoshi Esaki*** Ken Hirotsu**
D-serine is present at high levels in the mammalian brain including the central nervous system, higher than even some common amino acids, and is an endogenous ligand of the glycine site of N-methyl D-aspartate receptors. In glial cells, D-serine is synthesized by a serine racemase (SerR), a pyridoxal phosphate (PLP)-dependent enzyme enriched in astrocytes in mammalian brain, while its degradation is carried out by a D-amino acid oxidase. SerR has been shown previously to possess two distinct catalytic activities. In addition to the conversion of L-serine to D-serine, SerR catalyzes the alpha, beta-elimination of water from both stereoisomers of serine. It was reported that ATP together with magnesium is a physiological activator and enhances the racemase activity by tenfold. S. pombe gene homologous (sSerR) to mouse SerR was overexpressed in E. coli. The enzyme was purified and crystallized. Crystal structures of sSerR in its unliganded form, the complex with the activator analog (AMP-PCP) and the complex with serine in the closed form have been determined at 1.7, 1.9, and 1.7 Å resolution, respectively. The enzyme is a homodimer, and each subunit is divided into small and large domains. Superposition of the unliganded onto the closed forms reveals significant differences including a large conformational change at the domain level. The small domain rotates by about 20 degrees and approaches the large domain to close the active site. Interestingly, the substrate serine bound to the active site makes a covalent bond with PLP and the catalytic lysine and enhancer AMP-PCPs are bound to the subunit interface away from the active-site PLP.