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Latest revision as of 00:04, 22 October 2021

CYP6G1 fusioned with NADPH reductase

Usage and Biology

Enzyme CYP6G 1 is a class II cytochrome P450 monooxygenase native from the Drosophila melanogaster fly that has affinity for several xenobiotics as substrates including neonicotinoids and organophosphorus [2].

We aim to increase its enzyme catalytic efficiency by engineering the redox partner: the NADPH-dependent cytochrome P450 reductase (CPR), since the CYP6G1 enzyme belongs to type II and its enzymatic activity depends on the acquisition of electrons in an oxidation-reduction reaction that the CPR protein transfers from the cofactor NADPH. [4,7]

Inspired by the evolution of certain CYPs to become naturally “self-sufficient” such as cytochrome P450BM3 (CYP102A1) that belongs to class III [8] by the natural fusion of CYP with the cytochrome P450 reductase through a flexible linker [4] and the use of S. cerevisiae chassis as a eukaryotic expression system, we improved this part by artificially fusing CYP6G1 with the NADPH-dependent cytochrome P450 reductase by adding a flexible linker between the C-terminal CYP6G1 and N-terminal CPR sequence, respectively. Additionally, the codon of both coding sequences was optimized to be expressed in the Saccharomyces cerevisiae chassis, in order to obtain a correct expression of both enzymes in a single polypeptide chain.

Figure 1: Schematic representation of the P450-redox partner interaction. P450, cytochrome 450 monooxygenase; CPR, cytochrome P450 reductase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate.

Our improvement strategy in order to make CYP6G1 a self-sufficient cytochrome P450 monooxygenase enzyme carrying its own redox partner (CPR) we added a small flexible linker composed of amino acids (Gly-Gly-Gly-Gly-Ser) between the C-terminus of CYP6G1 and the N-terminus of cytochrome P450 reductase originally from Musca domestica.

Therefore, that linker containing small non-polar amino acids such as glycine (Gly) and polar residues such as serine (Ser) which gives the linker flexibility and stability in aqueous media, which in turn facilitates the movement and/or interaction of both proteins [1]. Additionally, in Figure 2, we didactically show the scheme of the new CYP6G1-CPR by the artificial fusion of the linker. It can be seen that the N-terminal sequence of the membrane-anchoring CPR was removed in order to facilitate the movement of CPR fused to CYP6G1.

Figure 2: Schematic representation of the novel CYP6G1 fused to NADPH-dependent cytochrome P450 reductase (CPR) by the flexible linker (GGGGS)x5. The N-terminal sequence of the membrane-anchoring CPR was removed to support the protein movement and rising the electron transfer efficiency. CYP6G1, cytochrome P450 monooxygenase; CPR, cytochrome P450 reductase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NADPH, reduced nicotinamide adenine dinucleotide phosphate.

The artificial fusion of both parts through this linker reduces the molar ratio from 15:1 to 1:1 between CPR and CYP6G1, respectively [4]. This means a higher electron transfer efficiency from the NADPH cofactor to the CYP6G1 enzyme, promoting much higher enzymatic turnover rates compared to the unfused CYP6G1 [4]. Consequently, this high turnover rate allows the enzyme to efficiently catalyze (e.g., hydroxylate) a greater amount of the substrate (i.e, imidacloprid) thus improving the enzymatic catalysis of CYP6G1.

References

1. Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357-1369. https://doi.org/10.1016/j.addr.2012.09.039
2. Joußen, N., Heckel, D. G., Haas, M., Schuphan, I., & Schmidt, B. (2008). Metabolism of imidacloprid and DDT by P450 CYP6G1 expressed in cell cultures of Nicotiana tabacum suggests detoxification of these insecticides in Cyp6g1-overexpressing strains of Drosophila melanogaster, leading to resistance. Pest Management Science, 64(1), 65-73. https://doi.org/10.1002/ps.1472
3. Labade, C. P., Jadhav, A. R., Ahire, M., Zinjarde, S. S., & Tamhane, V. A. (2018). Role of induced glutathione-S-transferase from Helicoverpa armigera (Lepidoptera: Noctuidae) HaGST-8 in detoxification of pesticides. Ecotoxicology and Environmental Safety, 147, 612-621. https://doi.org/10.1016/j.ecoenv.2017.09.028
4. Li, Z., Jiang, Y., Guengerich, F. P., Ma, L., Li, S., & Zhang, W. (2020). Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications. Journal of Biological Chemistry, 295(3), 833-849. https://doi.org/10.1016/S0021-9258(17)49939-X
5. Sadeghi, S. J., & Gilardi, G. (2013). Chimeric P450 enzymes: Activity of artificial redox fusions driven by different reductases for biotechnological applications: Artificial Fusions of CYP Heme Domain with Different Reductases. Biotechnology and Applied Biochemistry, 60(1), 102-110. https://doi.org/10.1002/bab.1086
6. Shephard, E. A., Phillips, I. R., Bayney, R. M., Pike, S. F., & Rabin, B. R. (1983). Quantification of NADPH: Cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique. Biochemical Journal, 211(2), 333-340. https://doi.org/10.1042/bj2110333
7. Talmann, L., Wiesner, J., & Vilcinskas, A. (2017). Strategies for the construction of insect P450 fusion enzymes. Zeitschrift Für Naturforschung C, 72(9-10), 405-415. https://doi.org/10.1515/znc-2017-0041
8. Whitehouse, C. J. C., Bell, S. G., & Wong, L.-L. (2012). P450 BM3 (Cyp102a1): Connecting the dots. Chem. Soc. Rev., 41(3), 1218-1260. https://doi.org/10.1039/C1CS15192D

Sequence and Features