Two-dimensional NMR and All-atom Molecular Dynamics of Cytochrome P450 CYP119 Reveal Hidden Conformational Substates

Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substr...

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Bibliographic Details
Published in:The Journal of biological chemistry 2010-03, Vol.285 (13), p.9594-9603
Main Authors: Lampe, Jed N., Brandman, Relly, Sivaramakrishnan, Santhosh, de Montellano, Paul R. Ortiz
Format: Article
Language:English
Subjects:
Archaeal Proteins - chemistry
Binding Sites
Catalysis
Catalytic Domain
Computer Modeling
Crystallography, X-Ray - methods
Cytochrome P-450 Enzyme System - chemistry
Cytochrome P450
Lauric Acids - chemistry
Ligands
Magnetic Resonance Spectroscopy - methods
Methyltyrosines - chemistry
Models, Molecular
Mutation
NMR
Protein Conformation
Protein Structure
Protein Structure and Folding
Spectrophotometry - methods
Sulfolobus acidocaldarius
Sulfolobus acidocaldarius - enzymology
UV Spectroscopy
Xenobiotics
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Summary:Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [13C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.087593