title: Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli.
abstract: P450sca-2 is an industrially important enzyme that stereoselectively converts mevastatin into pravastatin. However, little information or engineering efforts have been reported for this enzyme or its redox partner. In this study, we successfully reconstituted the P450sca-2 activity in Escherichia coli by co-expression with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from the Pseudomonas putida cytochrome P450cam system. With an HPLC-based screening assay, random mutagenesis was applied to yield a mutant (R8-5C) with a pravastatin yield of the whole-cell biotransformation 4.1-fold that of the wild type. P450sca-2 wild-type and R8-5C were characterized in terms of mevastatin binding and hydroxylation, electron transfer, and circular dichroism spectroscopy. R8-5C showed an active P450 expression level that was 3.8-fold that of the wild type, with relatively smaller changes in the apparent k(cat)/K(M) with respect to the substrate mevastatin (1.3-fold) or Pdx (1.5-fold) compared with the wild type. Thus, the increase in the pravastatin yield of the whole-cell biotransformation primarily came from the improved active P450 expression, which has resulted largely from better heme incorporation, although none of the six mutations of R8-5C are located near the heme active site. These results will facilitate further engineering of this P450sca-2 system and provide useful clues for improving other hybrid P450 systems.
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, Beijing, P.R. 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: D001673:Biodegradation, Environmental; D057005:Bioengineering / Q000379:methods*; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D005288:Ferredoxins / Q000378:metabolism; D006900:Hydroxylation; D007218:Industrial Microbiology; D008148:Lovastatin / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D017035:Pravastatin / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005288:Ferredoxins; D011994:Recombinant Proteins; C012258:mevastatin; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System; D008148:Lovastatin; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase; D017035:Pravastatin
keywords: Cytochrome P450sca-2; Pravastatin; Random mutagenesis; Redox partner; Whole-cell biotransformation