Novel iron-sulfur containing NADPH-reductase from Nocardia farcinica IFM10152 and fusion construction with CYP51 lanosterol demethylase.
CYP51, a sterol 14α-demethylase, is one of the key enzymes involved in sterol biosynthesis and requires electrons transferred from its redox partners. A unique CYP51 from Nocardia farcinica IFM10152 forms a distinct cluster with iron-sulfur containing NADPH-P450 reductase (FprD) downstream of CYP51. Previously, sequence alignment of nine reductases from N. farcinica revealed that FprC, FprD, and FprH have an additional sequence at their N-termini that has very high identity with iron-sulfur clustered ferredoxin G (FdxG). To construct an artificial self-sufficient cytochrome P450 monooxygenase (CYP) with only FprD, CYP51, and iron-sulfur containing FprD were fused together with designed linker sequences. CYP51-FprD fusion enzymes showed distinct spectral properties of both flavoprotein and CYP. CYP51-FprD F1 and F2 in recombinant Escherichia coli BL21(DE3) catalyzed demethylation of lanosterol more efficiently, with k(cat) /K(m) values of 96.91 and 105.79 nmol/min/nmol, respectively, which are about 35-fold higher compared to those of CYP51 and FprD alone.
School of Chemical and Biological Engineering, Institute of Bioengineering, Seoul National University, Seoul, South Korea.
Kwon-Young
KY
Choi
Eun-Ok
EO
Jung
Da-Hye
DH
Jung
Bishnu Prasad
BP
Pandey
Nahum
N
Lee
Hyungdon
H
Yun
Hyung-Yun
HY
Park
Byung-Gee
BG
Kim
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007810:Lanosterol / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009615:Nocardia / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis; D058886:Sterol 14-Demethylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D007810:Lanosterol; D009249:NADP; D058886:Sterol 14-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase