ROLE OF RESIDUE-363 AND RESIDUE-206 IN CONVERSION OF CYTOCHROME-P450 2B1 FROM A STEROID 16-HYDROXYLASE TO A 15-ALPHA-HYDROXYLASE

Four double and four triple site-directed mutants of cytochrome P450 2 B1 were constructed, expressed in COS cells, and assayed for androsten edione and testosterone hydroxylation. The mutants combined a Val-363 --> Ala substitution with an Ile-114 --> Val or Ala substitution and/o r a Gly-478 --> Ala or Ser substitution. Each of the individual mutati ons enhances androgen 15 alpha-hydroxylation, and the appropriate comb ination of Val or Ala at position 114 with Ala or Ser at position 478 has recently been shown to convert P450 2B1 from an androstenedione an d testosterone 16 beta-hydroxylase to a 15 alpha-hydroxylase (Halpert, J. R., and He, Y.-A. (1993) J. Biol. Chem. 268, 4453-4457). All eight mutants containing the Val-363 --> Ala substitution preferentially hy droxylated androstenedione and testosterone in the 15 alpha-position a nd thus functionally resemble mouse P450 2A4. However, unlike P450 2A4 , various single and multiple 2B1 mutants at positions 114, 363, and 4 78 mainly hydroxylated progesterone in the 16 alpha- rather than 15 al pha-position. By combining the Ile-114 --> Ala substitution with a Phe -206 --> Leu mutation (corresponding to Ala-117 and Leu-209 in P450 2A 4), P450 2B1 was converted to a progesterone 15 alpha-hydroxylase with retention of testosterone 15 alpha-hydroxylase activity. These studie s document the importance of residues 363 and 206 in determining the s ubstrate specificity of P450 2B1 and strongly support the hypothesis t hat the judicious combination of a small number of discrete mutations can be used to confer new specificities on P450 enzymes. (C) 1994 Acad emic Press, Inc.