Structure of cytochrome P450eryF: Substrate, inhibitors, and model compounds bound in the active site

• Published in: Steroids 1997, Vol.62 (1), p.112-116
• Main Authors: Cupp-Vickery, Jill R., Poulos, Thomas L.
• Format: Article
• Language: English
• Subjects: Actinomyces - enzymology; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Camphor - metabolism; Crystallography, X-Ray; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Enzyme Inhibitors - metabolism; Enzymes and enzyme inhibitors; Erythromycin - metabolism; Fundamental and applied biological sciences. Psychology; Hydrogen Bonding; Ketoconazole - metabolism; Models, Molecular; Mutation; Oxidoreductases; Protein Conformation; Sequence Homology, Amino Acid; Substrate Specificity
• ISSN: 0039-128X; 1878-5867
• DOI: 10.1016/S0039-128X(96)00168-7

Much of our understanding of P450 reaction mechanisms derives from studies on P450cam, a bacterial camphor hydroxylase. P450cam has served as the model for understanding detailed structure/function relationships in mammalian P450 enzymes, which have not proved amenable to x-ray crystallographic techniques. To expand and improve the P450 model, we solved the structure of P450eryF, a cytochrome P450 involved in erythromycin biosynthesis. The overall structure of P450eryF is similar to that of P450cam, but differs in the exact positioning of several α-helices, which results in the enlargement of the substrate-binding pocket. P450eryF also differs from P450cam in having alanine in place of the highly conserved threonine residue in the active site. To assess the role of this alanine residue, two mutant forms of P450eryF and a substrate analog were examined. Our findings suggest that P450eryF has evolved an active site that utilizes the substrate to assist in catalysis. In addition, the enlarged substrate binding pocket of P450eryF enables P450eryF to bind certain steroid compounds and azole-based steroid hydroxylase inhibitors. Crystals have been obtained for P450eryF complexed with the antifungal drug ketoconazole, and the high-resolution structure has been determined.