Abstract
On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F and G helices of mitochondrial P450s 27A1 and 11A. We introduced substitutions at Phe-207, Ile-211, and Phe-215 within putative helix F and at Trp-235 and Tyr-238 within putative helix G in P450 27A1 and compared wild type and mutants with respect to catalytic activity, product pattern, substrate binding, formation of hydrogen peroxide, and interaction with redox partner. Results indicate that the mutated residues are important for delivery of the correctly oriented substrate to the P450 active site. The I211K and F215K mutations, for example, affected the regioselectivity of P450 27A1-dependent hydroxylation reactions and conferred the P450 capacity to cleave the C-C bond of the substrate during the catalytic cycle. Studies of P450 11A1 indicate that Phe-202 has functions similar to those of its counterpart in P450 27A1 (Phe-215). We propose that putative helices F and G form the sides of the substrate-access channel, thus providing the additional mechanism to control regioselectivity of hydroxylation in mitochondrial P450s.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Binding Sites / genetics
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Catalysis
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Cattle
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Cholestanetriol 26-Monooxygenase
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Cholesterol Side-Chain Cleavage Enzyme / chemistry
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Cholesterol Side-Chain Cleavage Enzyme / genetics
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Cholesterol Side-Chain Cleavage Enzyme / metabolism
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Cytochrome P-450 Enzyme System / chemistry
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Cytochrome P-450 Enzyme System / genetics
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Cytochrome P-450 Enzyme System / metabolism*
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Humans
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Hydrogen Peroxide / metabolism
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Hydroxylation
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Kinetics
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Mass Spectrometry
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Mitochondria / enzymology*
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Mitochondria / genetics
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Protein Structure, Secondary / genetics
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Steroid Hydroxylases / chemistry
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Steroid Hydroxylases / genetics
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Steroid Hydroxylases / metabolism*
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Subcellular Fractions / enzymology
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Substrate Specificity / genetics
Substances
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Recombinant Proteins
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Cytochrome P-450 Enzyme System
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Hydrogen Peroxide
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Steroid Hydroxylases
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CYP27A1 protein, human
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Cholestanetriol 26-Monooxygenase
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Cholesterol Side-Chain Cleavage Enzyme