Bishnu Prasad Pandey

Cloning, expression and characterization of CYP102D1, a self-sufficient P450 monooxygenase from Streptomyces avermitilis.

Among 33 cytochrome P450s (CYPs) of Streptomyces avermitilis, CYP102D1 encoded by the sav575 gene is naturally a unique and self‐sufficient CYP. Since the native cyp102D1 gene could not be expressed well in Escherichia coli, its expression was attempted using codon‐optimized synthetic DNA. The gene was successfully overexpressed and the recombinant CYP102D1 was functionally active, showing a Soret peak at 450 nm in the reduced CO difference spectrum. FMN/FAD isolated from the reductase domain showed the same fluorescence in thin layer chromatography separation as the authentic standards. Characterization of the substrate specificity of CYP102D1 based on NADPH oxidation rate revealed that it catalysed the oxidation of saturated and unsaturated fatty acids with very good regioselectivity, similar to other CYP102A families depending on NADPH supply. In particular, CYP102D1 catalysed the rapid oxidation of myristoleic acid with a kcat/Km value of 453.4 ± 181.5 μm−1·min−1. Homology models of CYP102D1 based on other members of the CYP102A family allowed us to alter substrate specificity to aromatic compounds such as daidzein. Interestingly, replacement of F96V/M246I in the active site increased catalytic activity for daidzein with a kcat/Km value of 100.9 ± 10.4 μm−1·min−1 (15‐fold).

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680.

Regiospecific 3′‐hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent Km and kcat values of CYP105D7 for daidzein were 21.83 ± 6.3 µM and 15.01 ± 0.6 min−1 in the presence of 1 µM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO‐difference spectra at 450 nm using the whole cell extract. When the whole‐cell reaction for the 3′‐hydroxylation reaction of daidzein was carried out with 100 µM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6‐fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3′,4′‐trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h. Biotechnol. Bioeng. 2010. 105: 697–704.

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 kcat/Km 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.

Identification of the specific electron transfer proteins, ferredoxin, and ferredoxin reductase, for CYP105D7 in Streptomyces avermitilis MA4680

It was previously proposed that regiospecific hydroxylation of daidzein at 3′-position is mediated by cytochrome P450 hydroxylase (CYP105D7) in the presence of putidaredoxin (CamB) and putidaredoxin reductase (CamA) as electron transfer proteins from Pseudomonas putida. The genome sequence of Streptomyces avermitilis MA4680 revealed 33 P450 (CYPs) with 6 ferredoxin reductases (Fprs) and 9 ferredoxins (Fdxs) as their putative electron transfer partner proteins. To identify right endogenous electron transfer proteins for CYP105D7 activity, in vitro reconstitution, gene disruption, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) mRNA expression profile analysis were examined. The most effective electron transfer proteins for CYP105D7 appear to be FdxH (SAV7470), which is located downstream to CYP105D7 as a cluster, and FprD (SAV5675). Throughout our overall analysis, we proposed that the primary electron transfer pathway for CYP105D7 follows as such NAD(P)H→FdxH→FprD→CYP105D7.

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.