Comparison of the 17α-hydroxylase/C17,20-lyase activities of porcine, guinea pig and bovine P450c17 using purified recombinant fusion proteins containing P450c17 linked to NADPH-P450 reductase

Manjunath S. Shet, Charles W. Fisher, Yves Tremblay, Alain Belanger, Alan J Conley, J. Ian Mason, Ronald W. Estabrook

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E. coli and purified for use in enzymatic assays to determine similarities and differences in 17α-hydroxylase and lyase activities. The fusion proteins were found to catalyze both the 17α-hydroxylation of progesterone (P4) and pregnenolone (P5) to 17α-hydroxylated P4 and P5 (17α-OH P4 and 17α-OH P5) followed by the C17,20-lyase reaction for the conversion of these C21-17α-hydroxylated steroids to C19-steroids (the C17,20-lyase reaction). These in vitro studies show that (a) porcine P450c17 possesses cytochrome b5 (b5)-stimulated C17,20-lyase activity that converts 17αOH-P4 to androstenedione (AD) but also converts 17α-OHP5 to dehydroepiandrosterone (DHEA); (b) guinea pig P450c17 possesses a b5-stimulated C17,20-lyase activity that converts 17α-OH P4 to AD but does not convert 17α-OH P5 to DHEA., and (c) bovine P450c17 possesses a b5-stimulated C17,20-lyase activity that converts 17α-OH P5 to DHEA but does not convert 17α-OH P4 to AD. Thus, the P450c17 of each species differs in its ability to catalyze in vitro the conversion of C21-steroids to C19-steroids. In addition, each P450c17 is capable of catalyzing additional hydroxylation reactions leading to low levels of 2α-, 6β-, 16- and 21-hydroxy-metabolites. Porcine P450c17 also catalyzes the b5-dependent synthesis of andien-β (androsta-5,16-dien- 3β-ol) from P5. When the amino acid sequences of the three P450c17s were aligned there was an approximate 50% variation in the alignment identity (227 differences in the sequences of 509 amino acids). Alignment did not permit the assignment of specific amino acids or domains to the observed differences in enzymatic activities.

Original languageEnglish (US)
Pages (from-to)289-307
Number of pages19
JournalDrug Metabolism Reviews
Volume39
Issue number2-3
DOIs
StatePublished - Apr 2007

    Fingerprint

Keywords

  • Co-expression
  • Flavodoxin
  • Hydroxylases
  • NADPH-P450 reductase
  • P450 fusion proteins
  • Steroid hydroxylations

ASJC Scopus subject areas

  • Pharmacology (medical)
  • Pharmacology

Cite this