Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation

Paul J. Loida, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

Site-directed mutants were constructed in cytochrome P-450cam to re-engineer the stereochemistry and coupling of ethylbenzene hydroxyiation. The reaction with wild-type (WT) enzyme produces one regioisomer 1-phenylethanol with 5% reduced nicotinamide adenine deoxyribonucleic acid to product conversion of and a ratio of 73:27 for the R and S enantiomers respectively. Ethyibenzene was modeled into the active site of WT P-450cam in a rigid mode and oriented to optimize either pro-R or pro-S hydrogen abstraction. Residues T101, T185 and V247 make extensive contacts with the substrate in the static complexes and were therefore chosen for site-directed mutagenesis. Single mutants T101M, V247A and V247M are more stereospedik producing 89,87 and 82% (R)-1-phenylethanol respectively. The coupling of the reaction is doubled for the single mutants T185L, T185F and V247M. In an effort to engineer increased stereospecificity and coupling into a single catalyst the T101M, T185F and V247M mutants were combined in a multiple mutant of P-450cam.This protein hydroxylates ethyibenzene resulting in an R:S ratio of 87:13 for the 1-phenylethanols and 13% coupling of reducing equivalents to product. The catalytic stereospecificity and stoichiometry with T101M-T185F-V247M does not represent a summation of the changes observed for the single mutants. A portion of the individual effects on substrate recognition produced by the single substitutions is either eliminated or degenerate within the triple mutant.

Original languageEnglish (US)
Pages (from-to)207-212
Number of pages6
JournalProtein Engineering, Design and Selection
Volume6
Issue number2
DOIs
StatePublished - Feb 1993

Keywords

  • Coupling
  • Cytochrome P450cam
  • Site-directed mutagenesis
  • Stereospecificity

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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