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

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase fro m Pseudomonas fluorescens was replaced by a stereochemical analog, whi ch is spontaneously formed from natural FAD in alcohol oxidases from m ethylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within se conds. Crystals of the enzyme-substrate complex of modified FAD-contai ning p-hydroxybenzoate hydroxylase diffract to 2.1 Angstrom resolution . The crystal structure provides direct evidence for the presence of a n 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 phen olate form of the substrate (pk(a) = 7.2). The substrate acts as an ef fector highly stimulating the rate of enzyme reduction by NADPH (k(red ) > 500 s(-1)). The oxidative part of the catalytic cycle of a-FAD-con taining p-hydroxybenzoate hydroxylase differs from native enzyme. Part ial 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.