title: Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain L-amino-acid dioxygenase from Burkholderia ambifaria AMMD.
abstract: Ferrous ion- and α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3-hydroxylation of N-substituted branched-chain L-amino acids, especially N-succinyl-L-leucine, coupled to the conversion of α-ketoglutarate to succinate and CO(2). SadA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X-rays to 2.4 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (V(M) = 2.1 Å(3) Da(-1), 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Qin
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.forename: Akira
authors.initials: A
authors.lastname: Nakamura
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: D000596:Amino Acids / Q000378:metabolism; D019117:Burkholderia / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression
chemical_list: D000596:Amino Acids; D049308:Dioxygenases