CDNA CLONING, HETEROLOGOUS EXPRESSION, AND CHARACTERIZATION OF MOUSE CYP2G1, AN OLFACTORY-SPECIFIC STEROID HYDROXYLASE

CYP2G1 is expressed specifically in the olfactory mucosa in rabbits an d Fats. In the present study, a full-length cDNA for mouse CYP2G1 was obtained using a PCR approach with RNA preparations from the olfactory mucosa of C57BL/6 mice. Sequence comparisons indicated that mouse CYP 2G1 is highly homologous in deduced amino acid sequence to rabbit (82. 4% identity) and rat CYP2G1 (94.9% identity). RNA blot and immunoblot analyses indicated that mouse CYP2G1 is expressed only in the olfactor y mucosa. The coding region of the mouse CYP2G1 cDNA was cloned into a baculoviral expression vector for heterologous production of the enzy me in cultured insect cells. Heterologously expressed mouse CYP2G1 was active in a reconstituted system toward testosterone and progesterone , producing all the major metabolites detected in olfactory microsomal reactions, including 15 alpha, 15 beta-, and 2 beta-hydroxytestostero ne from testosterone and two unidentified metabolites from progesteron e. Kinetic analysis indicated that mouse CYP2G1 has relatively high af finities toward the steroid substrates, with K-m values in the micromo lar range for both testosterone and progesterone. At a substrate conce ntration of 10 mu M, microsomes of olfactory mucosa had much higher tu rnover numbers toward testosterone and progesterone than hepatic micro somes, consistent with the olfactory-specific expression of a high-aff inity sex steroid hydroxylase. These findings will facilitate further molecular genetics studies on the biological function of CYP2G1 in a m ouse model. (C) 1997 Academic Press.