Site-directed mutagenesis as a tool for molecular modeling of cytochrome P450 2B1.
Prompted by our previous homology model of cytochrome P450 2B1 based on the 3-D structure of P450cam [Szklarz, G. D., Ornstein, R. L., & Halpert, J. R. (1994) J. Biomol. Struct. Dyn. 12, 61-78], we constructed 11 new site-directed mutants at positions 100, 111, 205, 209, 291, 477, and 480 and expressed the enzymes in Escherichia coli. The mutations at positions 209, 477, and 480 affected androstenedione and progesterone hydroxylation as predicted by the model. For example, the Ile-477-->Ala and Ile-480-->Ala mutants retained < or = 5% activity with androstenedione and progesterone but were active with benzphetamine, whereas the Leu-209-->Ala mutant catalyzed 21-hydroxylation of progesterone. Mutations at the other positions, i.e., 100, 111, 205, and 291, did not change enzyme activity, contrary to predictions. Therefore, an improved molecular model of cytochrome P450 2B1 was constructed. An alignment of the P450 2B1 sequence with P450 BM-3, P450cam, and P450terp was optimized using data from site-directed mutagenesis at 27 positions in various cytochromes P450 2B and docking of androstenedione into the active site of the known crystal structures. Because all three structures were found to be suitable templates for P450 2B1, the new model was formulated on the basis of the crystallographic coordinates of the three proteins using a consensus strategy, a modeling method based on distance geometry calculations. The new model provides a means to explain alterations in regio- and stereospecificity of steroid hydroxylation upon residue substitution at key amino acid positions, including positions 114, 206, 209, 290, 302, 363, 367, 477, 478, and 480 in P450 2B1.
Szklarz
G D
GD
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
He
Y A
YA
Halpert
J R
JR
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment / Q000379:methods*; D017421:Sequence Analysis; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase