Coding

Part:BBa_K3890004

Designed by: Beatriz Toledo Akiti   Group: iGEM21_USP-Brazil   (2021-09-30)
Revision as of 23:23, 21 October 2021 by Biakiti (Talk | contribs)


CYP6G1 fusioned with NADPH reductase


Improved part of CYP6G1 (https://parts.igem.org/Part:BBa_K1197013) fused to NADPH-dependent cytochrome P450 reductase from Musca domestica (https://parts.igem.org/Part:BBa_K1197014) by adding a flexible linker composed of amino acids Glycine and a Serine residue [(Gly-Gly-Gly-Gly-Ser)x5] [1], resulting in the expression of both different proteins together in a single polypeptide chain. The N-terminal membrane anchor sequence of the cytochrome P450 reductase (CPR) was removed, and the codon of the coding sequence of both proteins was optimized for S. cerevisiae to be tested in a eukaryotic expression system.

The cytochrome P450 reductase transfers two electrons from the NADPH cofactor to CYP6G1 via a redox reaction, thus allowing CYP6G1 to hydrolyze carbons 4/5 or 4,5 of the imidazoline ring of imidacloprid producing the water-soluble 4-hydroxy-imidacloprid, 5-hydroxy-imidacloprid or the 4, 5-hydroxy-imidacloprid metabolites [1]. The artificial fusion of the cytochrome P450 reductase to CYP6G1 would improve the enzyme's catalytic activity by converting more substrate into products by increasing the rate of enzyme turnover as a result of greater efficiency in electron transfer by cytochrome P450 reductase.


Usage and Biology

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, it is possible to increase the enzyme's catalytic efficiency by engineering the redox partner: the NAPDH-dependent cytochrome P450 reductase (CPR) [2,3]. This interaction is inefficient because, commonly, in the microsome the CPR is shared with 15 CYPs, so the interaction molar ratio remains to 1:15, respectively [4].

Thus, by adding 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 BBa_K1197014, we can make the CYP6G1 self-sufficient. Therefore, that linker with small non-polar amino acids, such as glycine (Gly), and polar residues, such as serine (Ser), gives it flexibility and stability in aqueous media, which facilitates the movement and/or interaction of both proteins [5].

Using the linker to artificially fuse of both parts through can decrease the molar ratio between CPR and the CYP from 15:1 to 1:1 [4]. With this, a higher electron transfer efficiency from the NADPH cofactor to the enzyme can happen, promoting higher enzymatic turnover rates, allowing the enzyme to efficiently catalyze a higher quantity of the substrate (i.e, imidacloprid). Therefore, it improves the enzymatic catalysis of CYP6G1 [2].

References

[1] 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

[2] 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

[3] 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

[4] 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

[5] 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



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1947
    Illegal BglII site found at 3291
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 711
    Illegal BsaI.rc site found at 341
    Illegal BsaI.rc site found at 3242
    Illegal SapI.rc site found at 1822


[edit]
Categories
Parameters
None