Difference between revisions of "Part:BBa K3890004"
 
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<partinfo>BBa_K3890004 short</partinfo>
 
<partinfo>BBa_K3890004 short</partinfo>
  
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== Usage and Biology ==
  
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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.
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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].  
  
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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.
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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]
  
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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.
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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.
  
  
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===Usage and Biology===
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[[File:T--USP-Brazil--IGEM-USP-Description-Fusionedcyp1.png|667px|thumb|center|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.]]
  
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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].
 
  
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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 [https://parts.igem.org/Part: 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].  
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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 [https://parts.igem.org/Part:BBa_K1197014 Musca domestica].
  
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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].
+
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.
  
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===References===
 
  
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[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
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[[File:T--USP-Brazil--IGEM-USP-Description-Fusionedcyp2.png|595px|thumb|center|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.]]
  
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[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
 
  
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[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
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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.
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[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
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[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
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 +
===References===
  
 +
# 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
 +
# 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
 +
# 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
 +
# 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
 +
# 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
 +
# 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
 +
# 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
 +
# 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
  
  

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


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