Development of Colorimetric HTS Assay of Cytochrome P450 for ortho-Specific Hydroxylation, and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity

Title
Development of Colorimetric HTS Assay of Cytochrome P450 for ortho-Specific Hydroxylation, and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity
Author(s)
최권영[최권영]정은옥[정은옥]윤형돈양영헌[양영헌]Romas J. Kazlauskas[Romas J. Kazlauskas]김병기[김병기]
Keywords
HIGH-THROUGHPUT ASSAY; LABORATORY EVOLUTION; DIRECTED EVOLUTION; P450(BM3) CYP102A1; MASS-SPECTROMETRY; P450 BM3; ACID; EPOXIDATION; MUTANTS; SYSTEM
Issue Date
201307
Publisher
WILEY-V C H VERLAG GMBH
Citation
CHEMBIOCHEM, v.14, no.10, pp.1231 - 1238
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 COH correlates with a linear relationship to O-dealkylation activity on chemically introduced methoxy group at the corresponding COH. 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 mgL-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.
URI
http://hdl.handle.net/YU.REPOSITORY/29409http://dx.doi.org/10.1002/cbic.201300212
ISSN
1439-4227
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