Requirement of phenylalanine 343 for the preferential delta 4-lyase versus delta 5-lyase activity of rat CYP17.

Site-directed mutagenesis of a domain (amino acids 343-348) within the conserved region of rat CYP17 was performed to investigate species-specific differences between rat and human/bovine delta 4-versus delta 5-lyase activity. This domain displays substantial deviations between the rat and human/bovine/pig sequences and includes Arg346, which is known to be essential for delta 4-lyase (Kitamura, M., Buczko, E., and Dufau, M. L. (1991) Mol. Endocrinol. 5, 1373-1380) and delta 5-lyase activities. Analysis of the delta 4-lyase activity of mutant rat CYP17 cDNA expressed in nonsteroidogenic COS-1 cells revealed that substitution of Phe at position 343 in the rat with Ile of the human/bovine sequence resulted in a reduction in delta 4-lyase activity to levels in the range of the delta 5-supported reaction. This Phe343-->Ile mutant CYP17 did not exhibit changes either in delta 5-supported lyase activity or in delta 4- and delta 5-hydroxylase activities. Substitution of Asn344, Ser347, and His348 in rat CYP17 with the corresponding bovine amino acids Ser, Asn, and Arg did not enhance this effect. Thus, the reduced activity of the bovine CYP17 delta 4-lyase reaction can be mimicked in part in the rat polypeptide by the substitution of Phe343 with the bovine counterpart, Ile. Unlike the bovine CYP17-catalyzed reaction, the rat Phe343-->Ile mutant exhibited a low level lyase activity (kcat) that did not discriminate between delta 4- and delta 5-substrates. These results suggest that the presence of Phe343 enhances the delta 4-supported lyase activity possibly through stabilization of a delta 4-specific interaction.