Milhim Mohammed, Gerber Adrian, Neunzig Jens, Hannemann Frank, Bernhardt Rita
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.
J Biotechnol. 2016 Aug 10;231:83-94. doi: 10.1016/j.jbiotec.2016.05.035. Epub 2016 May 26.
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 P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia 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 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, 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.
细胞色素P450（P450s）需要电子传递伙伴来催化底物转化。就生物技术方法而言，新型电子传递蛋白的阐明具有特殊意义，因为它们可以影响P450s的酶活性和特异性。在当前工作中，我们展示了一种来自巨大芽孢杆菌的新型可溶性NADPH依赖性双黄素还原酶的鉴定和表征，该酶对一种细菌（CYP106A1）和一种微粒体（CYP21A2）P450具有活性，因此，我们将其称为巨大芽孢杆菌细胞色素P450还原酶（BmCPR）。对该蛋白的序列分析显示，除了保守的FMN、FAD和NADPH结合基序外，还存在带负电荷的簇，该簇被认为代表与P450s和/或细胞色素c的相互作用域。BmCPR在大肠杆菌中表达并纯化至同质。纯化的BmCPR呈现出特征性的双黄素还原酶光谱，并表现出细胞色素c还原活性。此外，在体外重组系统中，BmCPR能够分别支持CYP106A1和CYP21A2对睾酮和孕酮的羟基化。此外，鉴于其生物技术应用，BmCPR非常有前景，因为它可以成功用于建立基于CYP106A1和CYP21A2的全细胞生物转化系统，该系统分别在8小时内产生0.3g/L羟基睾酮产物和在6小时内产生0.16g/L 21-羟基孕酮。总之，本文报道的BmCPR在进一步的应用和研究中具有巨大潜力，在细菌和/或微粒体CYP依赖性生物转化中应予以考虑。
