Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation.
Site-directed mutants were constructed in cytochrome P-450cam to re-engineer the stereochemistry and coupling of ethylbenzene hydroxylation. 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. Ethylbenzene 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 stereospecific 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 ethylbenzene 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.
Loida
P J
PJ
Department of Biochemistry, University of Illinois, Urbana 61801.
Sligar
S G
SG
D001555:Benzene Derivatives / Q000378:metabolism; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D013379:Substrate Specificity
D001555:Benzene Derivatives; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase; C004912:ethylbenzene