title: Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer.
abstract: Hybrid P450 systems in which P450 monooxygenases are reconstituted with non-native or surrogate redox partners have become important for the engineering of this class of versatile enzymes. P450sca-2 from Streptomyces carbophilus stereoselectively hydroxylates mevastatin to yield pravastatin, a cholesterol-lowering drug. While S. carbophilus has been successfully applied in the industrial biotransformation process for pravastatin, the molecular study and engineering of P450sca-2 has been very limited. We have previously established a functional P450sca-2/Pdx/Pdr hybrid system. In this study, on the basis of a more active P450sca-2 mutant (R8-5C), five sites located in the substrate binding pocket, substrate access entrance, and presumed Pdx interaction interface were rationally chosen, and systematically subjected to site-directed saturation mutagenesis (SDSM), and three rounds of iterative saturation mutagenesis (ISM). A best mutant (Variant III) was obtained, which showed a whole cell biotransformation activity (377.5 mg/L) and an overall apparent k(cat) (6.37 min⁻¹) that was 7.1- and 10.0-fold that of the starting template R8-5C, respectively. Kinetic characterization revealed that most of the improvements seen for the SDSM and ISM mutants came from enhanced overall electron transfer, with the two sites at the interface between P450sca-2 and Pdx (T119 and N363) being most critical. Our study underscores the important role of electron transfer in a hybrid P450 system, and also demonstrates the utility of ISM in optimizing the redox partner interface. This should facilitate engineering of this and other important hybrid P450 systems.
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Ba
authors.forename: Pan
authors.initials: P
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Duan
authors.forename: Zhanglin
authors.initials: Z
authors.lastname: Lin
mesh_terms: D001665:Binding Sites; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017035:Pravastatin / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C069505:mevastatin hydroxylase; D017035:Pravastatin
keywords: cytochrome P450sca-2; electron transfer; iterative saturation mutagenesis; pravastatin; redox partner; site-directed saturation mutagenesis