title: Purification and characterization of benzoate-para-hydroxylase, a cytochrome P450 (CYP53A1), from Aspergillus niger.
abstract: Benzoate-para-hydroxylase (CYP51A or BpH) and NADPH:cytochrome P450 reductase from the filamentous fungus Aspergillus niger were purified to apparent homogeneity, using an overproducing A. niger strain. This is the first membrane-bound fungal cytochrome P450 to be isolated and characterized. Combining BpH with NADPH:cytochrome P450 oxidoreductase in the presence of the phospholipid dilauryl phosphatidylcholine restored the BpH activity, although to only a minor extent. Spectral analysis of BpH showed characteristic spectra for a cytochrome P450. Substrate binding studies with purified BpH as a function of temperature and as a function of pH were performed. Temperature-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 to the benzoate-BpH system. Two equilibrium constants were determined, K(1) for substrate binding without a spin change and K(2) for the spin change of the 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 break salt bridges at the binding site on the outside of the protein. pH-dependent kinetic studies with microsomes showed that the apparent K(M) values followed the trend observed for benzoate binding to purified BpH, while k(cat) values were virtually constant between pH 6.6 and 8.0 and decreased above pH 8, probably due to loss of productive interaction between BpH and NADPH:cytochrome P450 oxidoreductase. Research into the substrate specificity of 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 positions with specific, not too large groups. Substitution always leads to a lower affinity of the substrate. With one exception, all substrates were converted to their 4-hydroxy derivative. The exception, 3-methoxybenzoate, was demethylated to yield 3-hydroxybenzoate only. The restricted number of substrates and the specificity in catalysis suggest that BpH is not a general-purpose hydroxylase but that its role is confined to benzoate hydroxylation in the beta-ketoadipate pathway of A. niger.
authors.affiliation: Department of Microbiology and Enzymology, Delft University of Technology, Delft, The Netherlands. bfaber@chem.vu.nl
authors.forename: B W
authors.initials: BW
authors.lastname: Faber
authors.forename: R F
authors.initials: RF
authors.lastname: van Gorcom
authors.forename: J A
authors.initials: JA
authors.lastname: Duine
mesh_terms: D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology*; D050565:Benzoate 4-Monooxygenase; D019817:Benzoic Acid / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D007527:Isoenzymes; D010713:Phosphatidylcholines; C015153:1,2-dilauroylphosphatidylcholine; D019817:Benzoic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050565:Benzoate 4-Monooxygenase; D009251:NADPH-Ferrihemoprotein Reductase