Properties of a membrane-associated benzoate-4-hydroxylase from Rhodotorula graminis

Membrane fractions of benzoate-induced Rhodotorula graminis hydroxylated benzoate in the para position as demonstrated by high-performance liquid chromatography and isotopic thin-layer chromatography. Benzoate-4-hydroxylase activity was linear as a function of enzyme concentration (washed membranes) and time, and exhibited a pH optimum of 7.6. The enzyme utilized NADPH as a source of reducing equivalents, and was stimulated by FAD. The Km's for benzoate and NADPH were calculated as approximately 2.9 X 10(-5) M and approximately -1.9 X 10(-5) M, respectively. The particulate nature of benzoate-4-hydroxylase together with the fact that the enzyme was pteridine-independent indicates that it is distinct from the isofunctional enzyme previously described in filamentous fungi.