Interconversion of the androstenedione hydroxylase specificities of cytochromes p450 2B4 and 2B5 upon simultaneous site-directed mutagenesis of four key substrate recognition residues

被引:29
作者
He, YQ [1 ]
Szklarz, GD [1 ]
Halpert, JR [1 ]
机构
[1] UNIV ARIZONA,COLL PHARM,DEPT PHARMACOL & TOXICOL,TUCSON,AZ 85721
关键词
cytochrome P450; mutagenesis; steroid hydroxylation; structure-function relationships; heterologous expression; molecular modeling;
D O I
10.1006/abbi.1996.0493
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 15 alpha- and 16 alpha-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 16 beta-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 17 beta-acetyl group on progesterone as opposed to the oxo group on androstenedione may lead to interaction with additional amino acid residues.
引用
收藏
页码:152 / 160
页数:9
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