title: Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.
abstract: The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: W J
authors.initials: WJ
authors.lastname: van Berkel
authors.forename: M H
authors.initials: MH
authors.lastname: Eppink
authors.forename: H A
authors.initials: HA
authors.lastname: Schreuder
mesh_terms: D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry*; D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000302:isolation & purification; D005656:Fungal Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010843:Pichia / Q000201:enzymology; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013053:Spectrophotometry; D013237:Stereoisomerism
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D005182:Flavin-Adenine Dinucleotide; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D015109:4-Hydroxybenzoate-3-Monooxygenase