Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation |
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Authors: | Loida Paul J; Sligar Stephen G |
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Affiliation: | Department of Biochemistry, University of Illinois Urbana, Illinois, IL 61801, USA |
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Abstract: | Site-directed mutants were constructed in cytochrome P-450camto re-engineer the stereochemistry and coupling of ethylbenzenehydroxyiation. The reaction with wild-type (WT) enzyme producesone regioisomer 1-phenylethanol with 5% reduced nicotinamideadenine deoxyribonucleic acid to product conversion of and aratio of 73:27 for the R and S enantiomers respectively. Ethyibenzenewas modeled into the active site of WT P-450cam in a rigid modeand oriented to optimize either pro-R or pro-S hydrogen abstraction.Residues T101, T185 and V247 make extensive contacts with thesubstrate in the static complexes and were therefore chosenfor site-directed mutagenesis. Single mutants T101M, V247A andV247M are more stereospedik producing 89,87 and 82% (R)-1-phenylethanolrespectively. The coupling of the reaction is doubled for thesingle mutants T185L, T185F and V247M. In an effort to engineerincreased stereospecificity and coupling into a single catalystthe T101M, T185F and V247M mutants were combined in a multiplemutant of P-450cam.This protein hydroxylates ethyibenzene resultingin an R:S ratio of 87:13 for the 1-phenylethanols and 13% couplingof reducing equivalents to product. The catalytic stereospecificityand stoichiometry with T101MT185FV247M does notrepresent a summation of the changes observed for the singlemutants. A portion of the individual effects on substrate recognitionproduced by the single substitutions is either eliminated ordegenerate within the triple mutant. |
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Keywords: | coupling/ cytochrome P450cam/ site-directed mutagenesis/ stereospecificity |
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