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Characterization of bi-functional CYP154 from Nocardia farcinica IFM10152 in the O-dealkylation and ortho-hydroxylation of formononetin

Paper ID Volume ID Publish Year Pages File Format Full-Text
17526 42675 2010 8 PDF Available
Title
Characterization of bi-functional CYP154 from Nocardia farcinica IFM10152 in the O-dealkylation and ortho-hydroxylation of formononetin
Abstract

Among the 27 cytochrome P450s (CYPs) of Nocardia farcinica IFM10152, three CYPs have been identified as having O-dealkylation catalytic activity. Of the two that encode CYP154 subfamilies, the one encoded by the nfa22930 gene showed distinct O-dealkylation and subsequent hydroxylation of formononetin. Firstly, formononetin was O-dealkylated into daidzein, which was subsequently mono-hydroxylated at the 3′-position of the B-ring into ortho-dihydroxy-isoflavone. Apparent kcat/Km values of CYP154 for the O-dealkylation of formononetin and the hydroxylation of daidzein were 3.57 and 1.84 μM−1 min−1, respectively. The dissociation constants of CYP154 based on spectral changes upon binding to each substrate were 5.16 and 3.11 μM, respectively. Homology modeling and docking simulation found that Thr247 is responsible for the 3′-position hydroxylation reaction by forming a hydrogen bond with the 4′-hydroxyl group of daidzein that forces the proton at the 3′-position to face the heme center. Site-directed mutagenesis of Thr247 to alanine drastically decreased the binding affinity for daidzein (9.73 μM) as well as 3′-position hydroxylation catalytic activity by 3 fold (0.48 μM−1 min−1).

Keywords
Cytochrome P450; CYP154; O-dealkylation; Hydroxylation; Nocardia farcinica IFM10152
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Characterization of bi-functional CYP154 from Nocardia farcinica IFM10152 in the O-dealkylation and ortho-hydroxylation of formononetin
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 47, Issue 7, 8 December 2010, Pages 327–334
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
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Any Questions? feel free to contact us