Molecular modeling reveals the possible importance of a carbonyl oxygen binding pocket for the catalytic mechanism of p-hydroxybenzoate hydroxylase.

Abstract

p-Hydroxybenzoate hydroxylase catalyzes the hydroxylation of an aromatic substrate and uses flavin as a cofactor. The reaction probably occurs via a flavin 4a-hydroperoxide intermediate. In this study the crystal structure of 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin, an analogue of the flavin 4a-hydroperoxide intermediate, was fitted to the active site in the crystal structure of the p-hydroxybenzoate hydroxylase-3,4-dihydroxybenzoate complex. This model of an important catalytic intermediate fitted very well in the active site of p-hydroxybenzoate hydroxylase. The most striking result was that whereas with the normal flavin, the 0-4 of the flavin ring makes only poor hydrogen bonds with the protein, with the flavin 4a-hydroperoxide analogue, the same 0-4 makes strong hydrogen bonds with the NH groups of Gly-46 and Val-47. These two NH groups form a carbonyl oxygen binding pocket which has a geometry almost identical to the oxyanion hole found in several proteases. The possible consequences of this model for the reaction mechanism of p-hydroxybenzoate hydroxylase are discussed.