Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae.
Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
K M
KM
Kedzie
R M
RM
Philpot
J R
JR
Halpert
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D002021:Buffers; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D015151:Immunoblotting; D008163:Luminescent Measurements; D008862:Microsomes, Liver / Q000201:enzymology; D010957:Plasmids; D011374:Progesterone / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011817:Rabbits; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D002021:Buffers; D000735:Androstenedione; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System