title: Crystal structure of a novel N-substituted L-amino acid dioxygenase from Burkholderia ambifaria AMMD.
abstract: A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Qin
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.forename: Min Ze
authors.initials: MZ
authors.lastname: Jia
authors.forename: Akira
authors.initials: A
authors.lastname: Nakamura
authors.forename: Jun
authors.initials: J
authors.lastname: Ohtsuka
authors.forename: You-Lin
authors.initials: YL
authors.lastname: Xue
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawashima
authors.forename: Takuya
authors.initials: T
authors.lastname: Kasahara
authors.forename: Makoto
authors.initials: M
authors.lastname: Hibi
authors.forename: Jun
authors.initials: J
authors.lastname: Ogawa
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D019117:Burkholderia / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism; D006900:Hydroxylation; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010649:Phenylalanine / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D010649:Phenylalanine; D049308:Dioxygenases; D007930:Leucine