Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450s
Introduction of aromatic amino acids in electron transfer pathways yielded improved catalytic performance of cytochrome P450sCopyright: © Biotec
Meng, S., Li, Z., Ji, Y., Ruff, A. J., Liu, L., Davari, M. D., Schwaneberg, U., Chinese Journal of Catalysis , doi.org/10.1016/S1872-2067(23)64445-6
Cytochrome P450s are versatile catalysts for biosynthesis applications. In P450s reactions, two electrons are required to reduce the ferric heme iron and activate the subsequent reductions through electron transfer pathways (eTPs), which often represent the rate-limiting step in reactions. Hence, an in-depth understanding of the P450 ET pathway could increase our knowledge of these oxidases and benefit from their further applications.
The electron transfer efficiency of P450s are empowered by introducing aromatic amino acids.
In this study, the P450 BM3 from Bacillus megaterium was engineered for improved catalytic performance by redesigning proposed eTPs. By introducing aromatic amino acids on eTPs of P450 BM3, the “best” variant P2H02 (A399Y/Q403F) showed 13.9-fold improved catalytic efficiency (kcat /KM = 913.5 s-1 M-1 ) compared with P450 BM3 WT. Molecular dynamics simulations and electrons hopping pathways analysis revealed that aromatic amino acid substitutions bridging cofactor flavin mononucleotide and heme iron could increase electron transfer rates and, therefore, improve catalytic performance. Moreover, introducing tyrosines surprisingly showed positive effects on catalytic efficiency by protecting P450s from potential oxidative damage. Furthermore, the variant P2H02 showed improved catalytic efficiency (from 3.8- to 7.1-fold) towards five substrates (indole, benzo-1,4-dioxane, pseudocumene, CTAB, and p-xylene) with varying size and electronic properties. The general applicability of the electron transfer design through aromatic substitutions was also demonstrated by introducing the Q379Y substitution in CYP116B3 (9.16-fold improved catalytic efficiency). In summary, the engineering of eTPs by aromatic amino acid substitutions represents a powerful approach to design catalytically efficient P450s and could be expanded to other oxidases relying on long-range electron transfer pathways. Shuaiqi Meng was supported by a Ph.D. scholarship from the China Scholarship Council (CSC No. 201906880011).
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Aromatic amino acid substitutions bridge the cofactor flavin mononucleotide (FMN) and heme, improving electron transfer efficiency and catalytic performance of engineered P450s.