Purification and characterization of benzoate-para-hydroxylase, a cytochrome P450 (CYP53A1), from Aspergillus niger

Benzoate-para-hydroxylase (CYP51A or BpH) and NADPH:cytochrome P450 reducta se from the filamentous fungus Aspergillus niger were purified to apparent homogeneity, using an overproducing A. niger strain. This is the first memb rane-bound fungal cytochrome P450 to be isolated and characterized. Combini ng BpH with NADPH:cytochrome P450 oxidoreductase in the presence of the pho spholipid dilauryl phosphatidylcholine restored the BpH activity, although to only a minor extent. Spectral analysis of BpH showed characteristic spec tra for a cytochrome P450. Substrate binding studies with purified BpH as a function of temperature and as a function of pH were performed. Temperatur e-dependent studies, at pH 8.0, showed that the simplified spin equilibrium model originally proposed for camphor binding to cytochrome P450cam. (M. T . Fisher and S. G. Sligar, 1987, Biochemistry 26, 4797-4803) also applies t o the benzoate-BpH system. Two equilibrium constants were determined, K-1 f or substrate binding without a spin change and K-2 for the spin change of t he benzoate-BpH complex. pH-dependent binding studies showed that both K-1 and K-2 increase with pH, indicative of a higher affinity. As K-1 decreases more strongly with pH than K-2, we suggest that benzoate first binds to a binding site on the outside of the protein in a pH-dependent way, followed by transfer to the inside of the protein causing a spin change at the heme iron. The strong pH dependence of K-1 could be the result of the need to br eak salt bridges at the binding site on the outside of the protein. pH-depe ndent kinetic studies with microsomes showed that the apparent K-M values f ollowed the trend observed for benzoate binding to purified BpH, while k(ca t) values were virtually constant between pH 6.6 and 8.0 and decreased abov e pH 8, probably due to loss of productive interaction between BpH and NADP H:cytochrome P450 oxidoreductase. Research into the substrate specificity o f BpH showed that BpH can only use benzoic acid and some of its derivatives . Monosubstitution on the phenyl ring is allowed but only at certain positi ons with specific, not too large groups. Substitution always leads to a low er affinity of the substrate. With one exception, all substrates were conve rted to their 4-hydroxy derivative. The exception, 3-methoxybenzoate, was d emethylated to yield 3-hydroxybenzoate only. The restricted number of subst rates and the specificity in catalysis suggest that BpH is not a general-pu rpose hydroxylase but that its role is confined to benzoate hydroxylation i n the beta -ketoadipate pathway of A. niger. (C) 2001 Academic Press.