Interconversion of the androstenedione hydroxylase specificities of cytochromes P450 2B4 and 2B5 upon simultaneous site-directed mutagenesis of four key substrate recognition residues.
Based on recent studies of single reciprocal mutants of cytochrome P450 2B4 and the highly related P450 2B5 at positions 114, 294, 363, and 367 [G. D. Szklarz, Y. Q. He, K. M. Kedzie, J. R. Halpert, and V. L. Burnett (1996) Arch. Biochem. Biophys. 327,308-318], a number of multiple mutants were constructed, expressed in Escherichia coli, and assayed with androstenedione, progesterone, and benzyloxyresorufin. Simultaneous substitutions of Ile-114, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 with Phe, Thr, Val, and Ala, respectively from 2B5, resulted in a marked increase in androstenedione 15alpha- and 16alpha-hydroxylation compared with the wild-type enzyme and yielded a metabolite profile indistinguishable from that of cytochrome P450 2B5. Likewise, the reciprocal P450 2B5 quadruple mutant exhibited the specificity for 16beta-hydroxylation characteristic of the 2B4 wild type. The two reciprocal quadruple mutants of P450 2B4 and 2B5 also displayed benzyloxyresorufin dealkylase activities similar to those of the wild-type P450 2B5 and 2B4, respectively. However, the progesterone metabolite profile of P450 2B5 was not identical to that of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue. Therefore, the 17beta-acetyl group on progesterone as opposed to the oxo group on androstenedione may lead to interaction with additional amino acid residues.
He
Y Q
YQ
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
Szklarz
G D
GD
Halpert
J R
JR
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010078:Oxazines / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D010078:Oxazines; D011994:Recombinant Proteins; D000735:Androstenedione; D011374:Progesterone; C057754:benzyloxyresorufin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; C090279:cytochrome P-450 CYP2B5 (rabbit); C050356:steroid 15-alpha-hydroxylase