The rat 17α-hydroxylase-17,20-desmolase (CYP17) active site: Computerized homology modeling and site directed mutagenesis

• Published in: The Journal of steroid biochemistry and molecular biology 1995-03, Vol.52 (3), p.209-218
• Main Authors: Buczko, Ellen, Koh, YoungChul, Miyagawa, Yasushi, Dufau, Maria L.
• Format: Article
• Language: English
• Subjects: Aldehyde-Lyases - chemistry; Aldehyde-Lyases - genetics; Aldehyde-Lyases - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; Computer Simulation; Conserved Sequence - genetics; Cortisone Reductase - chemistry; Cortisone Reductase - genetics; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Enzymes and enzyme inhibitors; Exons - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Microsomes - enzymology; Models, Chemical; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidoreductases; Pregnenes - metabolism; Protein Structure, Secondary; Rats; Sequence Homology, Amino Acid; Steroid 17-alpha-Hydroxylase; Streptomyces - enzymology; Structure-Activity Relationship
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/0960-0760(94)00174-K

A homology model of the rat 17α-hydroxylase-17,20 desmolase (CYP17) † † CYP17 = P450c17; cytochrome P450 17 α ; 17α-hydroxylase-17,20 desmolase (or lyase). steroid binding domain was derived from the α/βF supersecondary structural element of the 3α/20β hydroxysteroid dehydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity. A CYP17 arginine-rich domain, including Arg346, Arg361 and Arg363, that has previously been shown to be important to CYP17 catalytic activity, is conserved in this HSD structural element between two HSD domains known to be important to C19 steroid binding. These two HSD motifs, in addition to a C-terminal domain at the apex of the steroid binding cavity, are also present in similar though not identical forms in the rat CYP17 sequence. The model was evaluated in terms of both hydroxylase/lyase activity and stability of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358, and Tyr431. Those amino acids located at folding junctions in the model steroid binding domain (Glu358, Arg361, and Tyr431) are each individually required to prevent degradation of the nascent protein, as well as for basic hydroxylase/lyase activity. Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a correlation exists between the length of exon 6 and the boundaries of the HSD supersecondary element. These studies demonstrate that exon 6 of the rat CYP17 is essential for CYP17 activity, and may be structurally related to the NAD-linked prokaryote α/βF supersecondary element.