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Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

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Design of enzyme fusions of BM3 4m and FDH2. (a) Detailed architecture and reaction scheme of the P450 BM3-FDH fusions as exemplified by the fusion construct with the enzyme order BF2 (real sizes of the enzymes are not considered). The formate dehydrogenase FDH2 (blue colored) catalyzes the oxidation of HCOO− to CO2 and reduction of NADP+ to NADPH. Cytochrome P450 consists of a BMP and BMR domain. The BMR domain (greenish colored) contains an FAD cofactor which partakes in the oxidation of NADPH to NADP+ and transfers two electrons to FMN, and an FMN cofactor which transfers the electrons (2 × e−) to the BMP domain (red colored). Subsequent substrate hydroxylation occurs at the active site of the BMP domain which contains heme and reduces O2 to water upon substrate hydroxylation. In the fusion enzyme, BM3 4m and FDH2 are bridged by a linker (white colored). (b) Schematic diagram for the constructed fusions with FDH2 (blue colored) positioned at the N-terminal and BM3 4m (red colored) positioned at the C-terminal (enzyme order F2B) and vice versa (enzyme order BF2). Short amino acid linkers are illustrated through the white box with the noted amino acids describing the composition of the linkers. The fusion constructs F2B and BF2 contain the methionine of the enzyme at the C-terminal position which was removed in the fusion constructs F2B-G1, F2B-G4 and F2B-P1.

Arsenij Kokorin successfully fused the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. The results recently appeared in Scientific Reports. Below you can read the abstract. The link to the whole publication can be found in the reference at the bottom.

"Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs."

Kokorin A, Parshin PD, Bakkes PJ, Pometun AA, Tishkov VI, Urlacher VB, 2021, Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity, Scientific Reports, 11(1):21706, https://doi.org/10.1038/s41598-021-00957-5

All publications of the biochemistry II can be found here!

Kategorie/n: BC Urlacher
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