title: Development of colorimetric HTS assay of cytochrome p450 for ortho-specific hydroxylation, and engineering of CYP102D1 with enhanced catalytic activity and regioselectivity.
abstract: A current challenge in high-throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid-agar plate-based HTS assay for screening ortho-specific hydroxylation of daidzein by sensing formaldehyde generated from the O-dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde-specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho-carbon position to COH correlates with a linear relationship to O-dealkylation activity on chemically introduced methoxy group at the corresponding COH. As a model system, a 4',7-dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B-ring, was examined for O-dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O-dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A-ring inactivated in order to find enhanced 3'-regioselectiviy). The whole-cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L(-1) production titer and greatly increased 3'-regioselectiviy (3'/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, 1 Kwanak-ro, 151-742 Seoul, South Korea.
authors.forename: Kwon-Young
authors.initials: KY
authors.lastname: Choi
authors.forename: Eun-Ok
authors.initials: EO
authors.lastname: Jung
authors.forename: Hyungdon
authors.initials: H
authors.lastname: Yun
authors.forename: Yung-Hun
authors.initials: YH
authors.lastname: Yang
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D002384:Catalysis; D003124:Colorimetry / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003640:Dealkylation; D057166:High-Throughput Screening Assays / Q000379:methods*; D006900:Hydroxylation; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D003577:Cytochrome P-450 Enzyme System
keywords: CYP102D1; HTS assay; O-dealkylation; cytochromes; diol-sensing; hydroxylation