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 Citations (Scopus)

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

Recombinant Fusion Proteins
NADPH-Ferrihemoprotein Reductase
Lyases
Mixed Function Oxygenases
Guinea Pigs
Swine
Dehydroepiandrosterone
Androstenedione
Steroids
Hydroxylation
Amino Acid Sequence
Conversion Disorder
Cytochromes b5
Amino Acids
Pregnenolone
Aptitude
Enzyme Assays
Cytochromes
Progesterone
Mammals

Keywords

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

ASJC Scopus subject areas

  • Pharmacology (medical)
  • Pharmacology

Cite this

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

In: Drug Metabolism Reviews, Vol. 39, No. 2-3, 04.2007, p. 289-307.

Research output: Contribution to journalArticle

Shet, Manjunath S. ; Fisher, Charles W. ; Tremblay, Yves ; Belanger, Alain ; Conley, Alan J ; Mason, J. Ian ; Estabrook, Ronald W. / 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. In: Drug Metabolism Reviews. 2007 ; Vol. 39, No. 2-3. pp. 289-307.
@article{c75a7aae6678476e91eaf507d44cb6bc,
title = "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",
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.",
keywords = "Co-expression, Flavodoxin, Hydroxylases, NADPH-P450 reductase, P450 fusion proteins, Steroid hydroxylations",
author = "Shet, {Manjunath S.} and Fisher, {Charles W.} and Yves Tremblay and Alain Belanger and Conley, {Alan J} and Mason, {J. Ian} and Estabrook, {Ronald W.}",
year = "2007",
month = "4",
doi = "10.1080/03602530701468391",
language = "English (US)",
volume = "39",
pages = "289--307",
journal = "Drug Metabolism Reviews",
issn = "0360-2532",
publisher = "Informa Healthcare",
number = "2-3",

}

TY - JOUR

T1 - 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

AU - Shet, Manjunath S.

AU - Fisher, Charles W.

AU - Tremblay, Yves

AU - Belanger, Alain

AU - Conley, Alan J

AU - Mason, J. Ian

AU - Estabrook, Ronald W.

PY - 2007/4

Y1 - 2007/4

N2 - 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.

AB - 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.

KW - Co-expression

KW - Flavodoxin

KW - Hydroxylases

KW - NADPH-P450 reductase

KW - P450 fusion proteins

KW - Steroid hydroxylations

UR - http://www.scopus.com/inward/record.url?scp=34548407689&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34548407689&partnerID=8YFLogxK

U2 - 10.1080/03602530701468391

DO - 10.1080/03602530701468391

M3 - Article

VL - 39

SP - 289

EP - 307

JO - Drug Metabolism Reviews

JF - Drug Metabolism Reviews

SN - 0360-2532

IS - 2-3

ER -