Document Detail

Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: implications for substrate binding.
MedLine Citation:
PMID:  20815369     Owner:  NLM     Status:  MEDLINE    
A previous study [Dickmann, L., et al. (2004) Mol. Pharmacol. 65, 842-850] revealed some unusual properties of the R108H mutant of cytochrome P450 2C9 (CYP2C9), including elevated thermostability relative to that of CYP2C9, as well as a UV-visible absorbance spectrum that was indicative of nitrogenous ligation to the heme iron. In our study, size-exclusion chromatography and UV-visible absorbance spectroscopy of CYP2C9 R108H monomers demonstrated that nitrogen ligation is indeed intramolecular. Pulsed electron paramagnetic resonance of CYP2C9 R108H monomers showed that a histidine is most likely bound to the heme as previously hypothesized. An energy-minimized model of the R108H mutant maintained a CYP fold, despite substantial movement of several loop regions of the mutant, and, therefore, represents an extreme example of a closed conformation of the enzyme. Molecular dynamics (MD) simulations of CYP2C9 were performed to study the range of energetically accessible CYP2C9 conformations. These in silico studies showed that the B-C loop region of CYP2C9 moves away from the heme to a position resembling the putative open conformation described for rabbit CYP2B4. A model involving the movement of the B-C loop region and R108 between the open and closed conformations of CYP2C9 is presented, which helps to explain the enzyme's ability to regio- and stereospecifically metabolize some ligands while allosterically activating others.
Arthur G Roberts; Matthew J Cheesman; Andrew Primak; Michael K Bowman; William M Atkins; Allan E Rettie
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2010-09-21
Journal Detail:
Title:  Biochemistry     Volume:  49     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2010 Oct 
Date Detail:
Created Date:  2010-12-14     Completed Date:  2011-01-19     Revised Date:  2014-09-22    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  8700-8     Citation Subset:  IM    
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MeSH Terms
Aryl Hydrocarbon Hydroxylases / chemistry,  genetics*,  metabolism*
Chromatography, Gel
Electron Spin Resonance Spectroscopy
Escherichia coli / genetics
Heme / metabolism*
Histidine / metabolism
Molecular Dynamics Simulation
Mutant Proteins / chemistry,  genetics,  metabolism
Protein Binding
Protein Conformation
Spectrophotometry, Ultraviolet
Substrate Specificity
Grant Support
Reg. No./Substance:
0/Mutant Proteins; 42VZT0U6YR/Heme; 4QD397987E/Histidine; EC Hydrocarbon Hydroxylases; EC protein, human

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