Title
A Novel NADPH-dependent flavoprotein reductase fromBacillus megaterium acts as an efficient cytochrome P450 reductase
Author
Mohammed Milhim, Adrian Gerber, Jens Neunzig, Frank Hannemann, Rita Bernhardt
Year
2016
Journal
Journal of Biotechnology
Abstract
Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450 s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in E. coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3 g/L hydroxy-testosterone products within 8 h and 0.16 g/L 21-hydroxyprogesterone within 6 h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Protein denaturation, Thermal stability, Thermodynamics, Protein folding, Biochemistry