Engineering of daidzein 3 '-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450

Title
Engineering of daidzein 3 '-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450
Author(s)
최권영[최권영]정은옥[정은옥]정다혜[정다혜]안벼리[안벼리]판데이[판데이]윤형돈송창민[송창민]박형연[박형연]김병기[김병기]
Keywords
STREPTOMYCES-AVERMITILIS; REGIOSELECTIVE HYDROXYLATION; BACILLUS-MEGATERIUM; RHODOCOCCUS SP; EXPRESSION; CLONING; MONOOXYGENASE; SUBTILIS
Issue Date
201206
Publisher
BIOMED CENTRAL LTD
Citation
MICROBIAL CELL FACTORIES, v.11
Abstract
A cytochrome P450 (CYP) enzyme, 3'-daidzein hydroxylase, CYP105D7 (3'-DH), responsible for daidzein hydroxylation at the 3'-position, was recently reported. CYP105D7 (3'-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3'-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a k(cat)/K-m value of 16.8 mu M-1 min(-1), which was about 24-fold higher than that of the 3'-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3'-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type.
URI
http://hdl.handle.net/YU.REPOSITORY/28158http://dx.doi.org/10.1186/1475-2859-11-81
ISSN
1475-2859
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