Alterations of the regiospecificity of progesterone metabolism by the mutagenesis of two key amino acid residues in rabbit cytochrome P450 2C3v.
A Ser/Thr difference at position 364 underlies a phenotypic difference between naturally occurring variants of microsomal cytochrome P450 2C3 in their capacity to catalyze the 6 beta-hydroxylation of progesterone, as well as in their sensitivity to the inhibitor 16 alpha-methyl-progesterone. Position 364 of P450 2C3 maps to a substrate contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450,101. In this study, Thr-364 of P450 2C3v, a progesterone 6 beta- and 16 alpha-hydroxylase, was replaced by Gly, Asp, Asn, Val, Leu, or Ile. The latter three amino acids did not alter the regiospecificity of P450 2C3v, whereas the Gly, Asp, and Asn substitutions each produced enzymes with properties that correspond closely to the Ser mutant that catalyzes 16 alpha-hydroxylation but not 6 beta-hydroxylation. The former are distinguished from the latter amino acids by their greater hydrophobicity and size. In contrast, the 16 alpha-hydroxylase activity could be greatly diminished by the introduction of an alanine replacement for Val-113. This mutation conferred progesterone 21- and 17 alpha-hydroxylase activity to P450 2C3v at the expense of 16 alpha-hydroxylase activity, leaving the 6 beta-hydroxylase activity largely unaffected. Compound mutants displayed the additive effects of the two mutations. These results are consistent with two distinct orientations for the binding of progesterone to P450 2C3v, resulting in 6 beta- and 16 alpha-hydroxylation, respectively. The binding of progesterone in these two orientations can be modulated relatively independently by modifications of the two key amino acid residues at 113 and 364.
Richardson
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TH
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Johnson
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EF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004789:Enzyme Activation; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011276:Pregnanediol / Q000494:pharmacology; D011374:Progesterone / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D000596:Amino Acids; D007527:Isoenzymes; D011994:Recombinant Proteins; C016836:allopregnane-3,20-diol; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450239:CYP2C3 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; C022657:steroid hormone 6-beta-hydroxylase; D011276:Pregnanediol