A hypervariable region of P450IIC5 confers progesterone 21-hydroxylase activity to P450IIC1.
Cytochrome P450IIC5 is a hepatic progesterone 21-hydroxylase while the 95% identical P450IIC4 has a greater than 10-fold higher Km for progesterone 21-hydroxylation and the 74% identical P450IIC1 does not hydroxylate progesterone at detectable rates. Previous work demonstrated that the apparent Km of P450IIC4 for progesterone 21-hydroxylation can be markedly improved by replacing a valine at position 113 with an alanine which is present at this position in P450IIC5. In the present studies, a single point mutation in cytochrome P450IIC1 that changed valine at position 113 to alanine conferred progesterone 21-hydroxylase activity to this enzyme. Although the catalytic activity was less than that of P450IIC5, these results indicate the residue 113 plays a critical role in the determination of the substrate/product selectivity in subfamily IIC P450s. By alignment with the sequence of P450cam, the segment of the polypeptide, residues 95-123, containing residue 113 corresponds to a substrate-contacting loop in the bacterial enzyme. The region containing residue 113, which is highly variable among family II P450s, may also be a substrate-contacting loop in the mammalian cytochromes P450. The exchange of this hypervariable region of cytochrome P450IIC1, residues 95-123, with that of P450IIC5 enhanced the 21-hydroxylase activity of the cells transfected with this chimera to levels similar to those of cells transfected with the plasmid encoding P450IIC5. Kinetic analysis of microsomes isolated from the transfected cells showed that the apparent Km for progesterone 21-hydroxylation of the chimera was indistinguishable from that of P450IIC5.(ABSTRACT TRUNCATED AT 250 WORDS)
Kronbach
T
T
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Kemper
B
B
Johnson
E F
EF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019475:Camphor 5-Monooxygenase; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D004247:DNA / Q000737:chemistry; D014644:Genetic Variation; D006900:Hydroxylation; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C065407:CYP2C5 protein, Oryctolagus cuniculus; D000072467:Cytochrome P450 Family 2; D013255:Steroid 21-Hydroxylase; D019475:Camphor 5-Monooxygenase