关键词: 细胞色素P450酶, 还原伴侣, MycG, RhFRED, 还原伴侣工程

Abstract:

Cytochrome P450 enzymes, which are widely involved in the key biosynthetic steps of many natural products, are considered as the most versatile biocatalysts in the nature. Redox partners responsible for electron transfer are indispensable for most P450 catalytic reactions. During the long evolution process, various P450-redox partner systems have been constituted by protein fusion and recombination. The protein-protein interaction and recognition between P450 and redox partner(s) are not only the key factor for functional reconstitution of P450s, but also can affect and change the catalytic functions and properties of P450 enzymes. To address the issue that how the choice and recombination of different redox partners affect the catalytic behaviors (reaction type, catalytic efficiency, product distribution, and electron transport) of the P450 enzyme, a number of fusion and separation P450 systems were constructed based on the two-domain redox partner protein RhFRED and P450 MycG. Specifically, the two domains of RhFRED were separated and expressed as stand-alone FMN and Fe₂S₂ proteins. The positions of these two domains and MycG were shuffled and engineered. In vitro biochemical reactions were carried out to explore the effect of different redox partner combinations on the MycG-catalyzed bioconversion of mycinamicin-Ⅳ (M-Ⅳ) and the electron transfer efficiency as well. As results, among the 20 tested catalytic systems, the functions of the multifunctional P450 enzyme MycG were successfully reconstituted through 12 combinations, in which three oxidative products including M-Ⅰ, M-Ⅱ and M-Ⅴ, and one demethylation product dMe-M-Ⅳ were produced. Besides, 4 combinations only led to the production of three oxidative products. In addition, no products were detected in the other 4 combinations. By simulating natural evolutionary strategy, the effects of three different catalytic systems of redox partner(s) and P450 enzymes, together with different protein organization forms on the functional modulating of MycG, were studied systematically. These results indicate that the P450 catalytic properties are affected and modulated by not only the identity, but also the position and organization of redox partner (s), probably via alternative protein-protein interactions.

Key words: cytochrome P450 enzymes, redox partners, MycG, RhFRED, redox partner engineering