CRYSTAL-STRUCTURE OF P-HYDROXYBENZOATE HYDROXYLASE RECONSTITUTED WITH THE MODIFIED FAD PRESENT IN ALCOHOL OXIDASE FROM METHYLOTROPHIC YEASTS - EVIDENCE FOR AN ARABINOFLAVIN

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 Angstrom 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-PAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pk(a) = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (k(red) > 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.