title: Crystallization and preliminary X-ray analysis of the oxygenase component (HpaB) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8.
abstract: The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase enzyme catalyzes the hydroxylation of 4HPA to 3,4-dihydroxyphenylacetate in the initial step of the degradation pathway of 4HPA. This enzyme consists of two components: an oxygenase (HpaB) and a reductase (HpaC). HpaB hydroxylates 4HPA using an oxygen molecule and a reduced flavin, which is supplied by HpaC. HpaB from Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized. Crystals of HpaB were grown in 0.4 M 1,6-hexanediol, 0.1 M sodium acetate pH 5.0 and 25% (v/v) glycerol and diffracted X-rays to a resolution of 1.60 A. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 91.8, b = 99.6, c = 131.1 A. The asymmetric unit volume provides space for only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient V(M) of 2.8 A3 Da(-1) and a solvent content of 55.1%. Platinum-derivatized crystals of HpaB were prepared by soaking native crystals in a solution containing 1 mM ammonium tetrachloroplatinate(II) for 1 d and diffracted X-rays to a resolution of 2.50 A. MAD data were successfully collected for structural determination using these crystals.
authors.affiliation: RIKEN SPring-8 Center, Koto 1-1-1, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Seong-Hoon
authors.initials: SH
authors.lastname: Kim
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Miyatake
authors.forename: Tamao
authors.initials: T
authors.lastname: Hisano
authors.forename: Wakana
authors.initials: W
authors.lastname: Iwasaki
authors.forename: Akio
authors.initials: A
authors.lastname: Ebihara
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D010648:Phenylacetates / Q000737:chemistry; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D029968:Escherichia coli Proteins; D010648:Phenylacetates; C026246:4-hydroxyphenylacetate; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase