Mechanistic insights into p-hydroxybenzoate hydroxylase from studies of the mutant Ser212Ala

In the crystal structure of native p-hydroxybenzoate hydroxylase, Ser212 is within hydrogen bonding distance (2.7 Angstrom) of one of the carboxylic o xygens of p-hydroxybenzoate. In this study, we have mutated residue 212 to alanine to study the importance of the serine hydrogen bond to enzyme funct ion, Comparisons between mutant and wild type (WT) enzymes with the natural substrate p-hydroxybenzoate showed that this residue contributes to substr ate binding. The dissociation constant for this substrate is 1 order of mag nitude higher than that of WT, but the catalytic process is otherwise uncha nged, When the alternate substrate, 2,4-dihydroxybenzoate, is used, two pro ducts are formed (2,3,4-trihydroxybenzoate and 2,4,5-trihydroxybenzoate), w hich demonstrates that this substrate can be bound in two orientations. Kin etic studies provide evidence that the intermediate with a high extinction coefficient previously observed in the oxidative half-reaction of the WT en zyme with this substrate is composed of contributions from both the dienone form of the product and the C4a-hydroxyflavin. During the reduction of the enzyme-2,4-dihydroxybenzoate complex by NADPH with 2,4-dihydroxybenzoate, a rapid transient increase in flavin absorbance is observed prior to hydrid e transfer from NADPH to FAD. This is direct evidence for movement of the f lavin before reduction occurs.