A comparative approach to structure-function studies of mammalian aromatases

Alan J Conley, Samantha Mapes, C. J. Corbin, Doug Greger, Karen Walters, John Trant, Sandra Graham

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

To date, structure-function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B′ and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.

Original languageEnglish (US)
Pages (from-to)289-297
Number of pages9
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume79
Issue number1-5
DOIs
StatePublished - 2001

Fingerprint

Aromatase
Etomidate
Isoenzymes
Enzymes
Cytochrome P-450 Enzyme System
Swine
Substrates
Enzyme activity
Metabolism
Assays
Point Mutation
Computer Simulation
Water
DNA

Keywords

  • Chimeric enzymes
  • P450arom
  • Structure-function studies

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

A comparative approach to structure-function studies of mammalian aromatases. / Conley, Alan J; Mapes, Samantha; Corbin, C. J.; Greger, Doug; Walters, Karen; Trant, John; Graham, Sandra.

In: Journal of Steroid Biochemistry and Molecular Biology, Vol. 79, No. 1-5, 2001, p. 289-297.

Research output: Contribution to journalArticle

Conley, Alan J ; Mapes, Samantha ; Corbin, C. J. ; Greger, Doug ; Walters, Karen ; Trant, John ; Graham, Sandra. / A comparative approach to structure-function studies of mammalian aromatases. In: Journal of Steroid Biochemistry and Molecular Biology. 2001 ; Vol. 79, No. 1-5. pp. 289-297.
@article{41ab2845010f42369b6f6a3f11a0b3dc,
title = "A comparative approach to structure-function studies of mammalian aromatases",
abstract = "To date, structure-function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B′ and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.",
keywords = "Chimeric enzymes, P450arom, Structure-function studies",
author = "Conley, {Alan J} and Samantha Mapes and Corbin, {C. J.} and Doug Greger and Karen Walters and John Trant and Sandra Graham",
year = "2001",
doi = "10.1016/S0960-0760(01)00145-5",
language = "English (US)",
volume = "79",
pages = "289--297",
journal = "Journal of Steroid Biochemistry and Molecular Biology",
issn = "0960-0760",
publisher = "Elsevier Limited",
number = "1-5",

}

TY - JOUR

T1 - A comparative approach to structure-function studies of mammalian aromatases

AU - Conley, Alan J

AU - Mapes, Samantha

AU - Corbin, C. J.

AU - Greger, Doug

AU - Walters, Karen

AU - Trant, John

AU - Graham, Sandra

PY - 2001

Y1 - 2001

N2 - To date, structure-function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B′ and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.

AB - To date, structure-function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B′ and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.

KW - Chimeric enzymes

KW - P450arom

KW - Structure-function studies

UR - http://www.scopus.com/inward/record.url?scp=0035714770&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035714770&partnerID=8YFLogxK

U2 - 10.1016/S0960-0760(01)00145-5

DO - 10.1016/S0960-0760(01)00145-5

M3 - Article

C2 - 11850235

AN - SCOPUS:0035714770

VL - 79

SP - 289

EP - 297

JO - Journal of Steroid Biochemistry and Molecular Biology

JF - Journal of Steroid Biochemistry and Molecular Biology

SN - 0960-0760

IS - 1-5

ER -