Nonsubstrate recognition site residues are involved in testosterone hydroxylation by cytochrome P450 CYP 2C11.
We have previously characterized an allelic variant of cytochrome P450 CYP 2C11 from the Gunn rat that differs at three positions (amino acids 4, 116, and 187) from the predominant allele from Wistar rats and that displays a dramatically reduced testosterone hydroxylation activity. To assess the relative contribution of these mutations to the decrease in the enzymatic activity we constructed single and double mutants and coexpressed them with reductase. Testosterone metabolism was determined with a baculovirus/insect cell expression system. None of the identified positions alone is critical for the activity since the reversion of one of these mutations is unable to restore fully the Wistar-type activity. The activity of CYP 2C11 containing either the Asn116Ser substitution or the Phe187Leu represents congruent with30% of the activity of the CYP 2C11 Wistar-type protein. In contrast, the activity of the Val4Ala mutated protein is only 10% that of the Wistar-type protein, close to that of the Gunn-type protein. This study reevaluates the contribution of amino acid 4 to the catalysis by cytochrome P450 2C11 and points out the role of extra SRS residues.
Biagini
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NIEHS-LST, MD F204, Research Triangle Park, North Carolina, 27709, USA.
Philpot
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Célier
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D015870:Gene Expression; D006900:Hydroxylation; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011911:Rats, Gunn; D017208:Rats, Wistar; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism*
D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase