THE RAT 17-ALPHA-HYDROXYLASE-17,20-DESMOLASE (CYP17) ACTIVE-SITE - COMPUTERIZED HOMOLOGY MODELING AND SITE-DIRECTED MUTAGENESIS

A homology model of the rat 17 alpha-hydroxylase-17,20 desmolase (CYP1 7)dagger steroid binding domain was derived from the alpha/beta F supe rsecondary structural element of the 3 alpha/20 beta hydroxysteroid de hydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity. A CYP17 arginine-rich doma in, including Arg346, Arg361 and Arg363, that has previously been show n to be important to CYP17 catalytic activity, is conserved in this HS D 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 mod el was evaluated in terms of both hydroxylase/lyase activity and stabi lity of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361 Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358 , and Tyr431. Those amino acids located at folding junctions in the mo del steroid binding domain (Glu358, Arg361, and Tyr431) are each indiv idually required to prevent degradation of the nascent protein, as wel l as for basic hydroxylase/lyase activity. Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a cor relation 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 alpha/beta F supersecondary elem ent.