title: Catalytic mechanism of serine racemase from Dictyostelium discoideum.
abstract: The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of D- and L-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to L-serine dehydrase; S81A showed no racemase activity and had significantly reduced D-serine dehydrase activity, but it completely retained its L-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove D-serine dehydration by abstracting the α-hydrogen in D-serine. Our data suggest that the abstraction and addition of α-hydrogen to L- and D-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Frou-chou, Chikusa, Nagoya, Aichi 464-8601, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Ito
authors.forename: Motoki
authors.initials: M
authors.lastname: Maekawa
authors.forename: Shuhei
authors.initials: S
authors.lastname: Hayashi
authors.forename: Masaru
authors.initials: M
authors.lastname: Goto
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hemmi
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoshimura
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D004023:Dictyostelium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019998:Racemases and Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000099300:Serine Racemase
chemical_list: D015800:Protozoan Proteins; D019998:Racemases and Epimerases; D000099300:Serine Racemase