Structure and mechanism of para-hydroxybenzoate hydroxylase

Abstract

Para-hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein involved in degradation of aromatic compounds, and it has become a model for enzymes involved in the oxygenation of a substrate. The chemical and kinetic mechanisms of this enzyme are described and integrated with an outline of the structure of the protein from crystallographic analysis. The structure is unusual because there is no recognizable domain for the binding of NADPH involved in the reaction. Recently, mechanistic studies of site-directed mutants, combined with structural analyses, have provided some exciting discoveries about protein function. The substrate during catalysis is largely isolated from solvent in the active site, a necessary condition for successful product formation. The flavin ring structure moves substantially in the active site, probably to enable substrate and product exchange into this site and possibly to regulate the reduction of the flavin by NADPH. A chain of H-bonds can connect p-hydroxy-benzoate in the active site of the enzyme with the protein surface. This chain is responsible for the reversible formation of substrate phenolate anion observed in the active site and partly responsible for the reactivity of this substrate.