Chimerogenesis in estimation of specificity and pathway directions for cytochrome p45017alpha catalyzed reactions.
Cytochrome P45017alpha is a key enzyme in steroid hormone biosynthesis. It catalyzes the reaction of 17alpha-hydroxylation of progesterone (P4) and pregnenolone (P5) and the 17,20-lyase reaction resulting in side chain cleavage of C21 steroids to form C19 steroids. Depending on the activity of cytochrome P45017alpha, steroid hormone biosynthesis pathways are directed either for biosynthesis of mineralocorticoids and glucocorticoids or sex hormones. The formation of sex hormones starts from biosynthesis of androstenedione. Androstenedione formation is a result of two reactions: 17,20-lyase reaction of 17alpha-hydroxyprogesterone (Delta4-pathway) and 3beta-hydroxysteroid dehydrogenase/Delta4,Delta5-isomerase reaction using dehydroepiandrosterone as substrate (Delta5-pathway). In case of exclusive direction of the 17,20-lyase reaction either through the Delta4- or the Delta5-pathway, the formation of sex hormones depends more on specificity and activity of 3beta-hydroxysteroid-dehydrogenase/Delta4,Delta5-isomerase. Depending on species, the cytochromes P45017alpha can utilize as a substrate for 17,20-lyase activity Delta4-steroids, Delta5-steroids, or both types of steroids. To identify the structural elements of cytochrome P45017alpha responsible for substrate recognition, in the present work we used exchange of homologous fragments of cytochrome P45017alpha having different types of activities. We engineered more than 10 different types of chimeric cytochrome P45017alpha. Chimeric cytochromes P45017alpha have been expressed in E. coli and purified. The expression of chimeric cytochrome P45017alpha with the point of exchange between exons III and IV results in inability of the recombinant hemeprotein to properly bind heme. The determination of activity of chimeric cytochromes P45017alpha shows that the structural element responsible for switching activity between Delta4- or Delta5-pathway is located in the region of polypeptide chain coded by exons II-V of CYP17 gene.
Gilep
A A
AA
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk 220141, Belarus.
Estabrook
R W
RW
Usanov
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006736:Horses; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D015398:Signal Transduction; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013552:Swine
D011993:Recombinant Fusion Proteins; D013254:Steroid 17-alpha-Hydroxylase