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 kcat/Km 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. Biotechnol. Bioeng. 2012; 109:630–636. © 2011 Wiley Periodicals, Inc. 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 E. coli BL21(DE3) catalyzed demethylation of lanosterol more efficiently, with kcat/Km values of 96.91 and 105.79 nmol/min−1/nmol, respectively, which are about 35‐fold higher compared to those of CYP51 and FprD alone.