Engineering of daidzein 3'-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450.
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 μM(-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.
School of Chemical and Biological Engineering, Institute of Bioengineering, Seoul National University, Seoul, South Korea.
Kwon-Young
KY
Choi
EunOk
E
Jung
Da-Hye
DH
Jung
Byeo-Ri
BR
An
Bishnu Prasad
BP
Pandey
Hyungdon
H
Yun
Changmin
C
Sung
Hyung-Yeon
HY
Park
Byung-Gee
BG
Kim
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006900:Hydroxylation; D007529:Isoflavones / Q000378:metabolism*; D007700:Kinetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D007529:Isoflavones; C004742:daidzein; D003577:Cytochrome P-450 Enzyme System