title: Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization.
abstract: There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase.
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Lijun
authors.initials: L
authors.lastname: Liu
authors.forename: Kousuke
authors.initials: K
authors.lastname: Iwata
authors.forename: Akiko
authors.initials: A
authors.lastname: Kita
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kawarabayasi
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D019711:Pyrococcus / Q000201:enzymology*; D016415:Sequence Alignment
chemical_list: D011732:Pyridoxal Phosphate; D000591:Amino Acid Isomerases; C019749:aspartate racemase