Role of residue 480 in substrate specificity of cytochrome P450 2B5 and 2B11.
The role of residue 480 as a determinant of the specificities of cytochrome P450 2B5 and 2B11 toward androstenedione, progesterone, 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), and benzyloxyresorufin has been investigated. Two reciprocal mutants at position 480, 2B5 Val-480 -> Leu and 2B11 Leu -> Val, two hybrid enzymes, H1B (amino acid residues 1-370 from P450 2B11, 371-491 from P450 2B5) and H2B (amino acid residues 1-370 from P450 2B5, 372-494 from P450 2B11) (Kedzie et al., 1993, Biochim. Biophys. Acta 1164, 124-132), and two hybrid back mutants, H1B Val-480 -> Leu and H2B Leu-480 -> Val, were constructed and expressed in Escherichia coli, and compared with the wild-type enzymes P450 2B5 and 2B11. For androstenedione metabolism, the Leu-480 -> Val mutation in P450 2B11 resulted in an increase in the 16 beta-OH:16 alpha-OH ratio from 1.2 to 3, whereas the Val-480 -> Leu mutation in hybrid H1B decreased the 16 beta-OH:16 alpha-OH ration from 4.2 to 1.1 In the case of progesterone, the Leu-480 -> Val mutant of P450 2B11 displayed 3-fold higher 16 alpha- and 21-hydroxylase activities than the wild-type 2B11. In the absence of cytochrome b5, 2B11 L480V displayed half of the benzyloxyresorufin O-dealkylase (BROD) activity of 2B11 wild-type, whereas H1B V480L showed 5,6-fold higher activity than H1B. Therefore, residue 480 seems to play an important role in steroid and benzyloxyresorufin metabolism by P450 2B11. In contrast, the mutation Leu-480 -> Val did not have a significant effect on the 245-HCB hydroxylase activity of P450 2B11, and the mutation Val-480 -> Leu in P450 2B5 had no notable effect on its progesterone hydroxylase activity. Cytochrome b5 caused marked stimulation of the BROD activity of P450 2B11 and H1B and their mutants. Furthermore, P450 2B5 exhibited different progesterone metabolite profiles in the absence or presence of cytochrome b5.
Liu
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J
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721, USA.
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Y A
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Halpert
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D000595:Amino Acid Sequence; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D004285:Dogs; D004926:Escherichia coli; D007930:Leucine; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011078:Polychlorinated Biphenyls / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques; D014633:Valine
D017931:DNA Primers; D011994:Recombinant Proteins; D000735:Androstenedione; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D011078:Polychlorinated Biphenyls; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C090278:cytochrome P-450 CYP2B11 (dog); C090279:cytochrome P-450 CYP2B5 (rabbit); D007930:Leucine; D014633:Valine; C014024:2,4,5,2',4',5'-hexachlorobiphenyl