In silico Evaluation of Substrate Binding Site and Rare Codons in the Structure of CYP152A1

Author(s): Mojtaba Mortazavi, Navid Nezafat, Manica Negahdaripour, Mohammad J. Raee, Masoud Torkzadeh-Mahani, Ali Riahi-Madvar, Younes Ghasemi*

Journal Name: Current Proteomics

Volume 17 , Issue 1 , 2020

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Abstract:

Background: The Cytochromes P450 (CYPs) have an essential role in the oxidation of endogenous and exogenous molecules. The CYPs are identified in all domains of life, but the CYP152A1 from Bacillus subtilis is specially considered for clinical and industrial applications. The molecular cloning of a new type of CYP from Bacillus subtilis was reported, previously. Here, we describe the hidden layer of biological information of the CYP152A1 enzyme, which can help researchers for better understanding of enzyme application. In this study, four rare codons of enzyme, including Arg63, Arg187, Arg276, and Arg338 were identified and evaluated using the bioinformatics web servers.

Methods: Through in silico modeling of CYP152A1 via the I-TASSER server, the above-mentioned rare codons were studied in the structure of enzyme that may have an important role in the proper folding of CYP152A1. In the following, the substrate binding site of CYP152A1 was studied by AutoDock Vina, and the heme and palmitic acid were considered as the substrates.

Results: The results of docking study elucidated the Arg242 in the active site is closely related to the substrate binding site of CYP152A1, which help us to further clarify the mechanism of the enzyme reaction.

Conclusion: Studies of these hidden information’s can enhance our understanding of CYP152A1 folding and protein expression challenges. Moreover, identification of rare codons can help in the rational design of new and effective drugs.

Keywords: CYP152A1, Rare codon, AutoDock Vina, substrate docking, bioinformatics, kinetics.

[1]
Makris, T.; Denisov, I.; Schlichting, I.; Sligar, S.; de Montellano, P.O. In: Cytochrome P450: structure, mechanism, and biochemistry; Ed., Ortiz de Montellano, P.R. Plenum Publishers, NY, 2005, p. 149.
[2]
Sono, M.; Roach, M.P.; Coulter, E.D.; Dawson, J.H. Heme-containing oxygenases. Chem. Rev., 1996, 96, 2841-2888.
[3]
Zhao, Y.J.; Cheng, Q.Q.; Su, P.; Chen, X.; Wang, X.J.; Gao, W.; Huang, L.Q. Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants. Appl. Microbiol. Biotechnol., 2014, 98, 2371-2383.
[4]
Lamb, D.C.; Lei, L.; Warrilow, A.G.; Lepesheva, G.I.; Mullins, J.G.; Waterman, M.R.; Kelly, S.L. The first virally encoded cytochrome P450. J. Virol., 2009, 83, 8266-8269.
[5]
Hanukoglu, I. Electron transfer proteins of cytochrome P450 systems. Adv. Mol. Cell Biol., 1996, 14, 29-56.
[6]
Rittle, J.; Green, M.T. Cytochrome P450 compound I: Capture, characterization, and CH bond activation kinetics. Science, 2010, 330, 933-937.
[7]
Schwaneberg, U.; Appel, D.; Schmitt, J.; Schmid, R.D. P450 in biotechnology: Zinc driven ω-hydroxylation of p-nitrophenoxy-dodecanoic acid using P450 BM-3 F87A as a catalyst. J. Biotechnol., 2000, 84, 249-257.
[8]
Pazmino, D.T.; Winkler, M.; Glieder, A.; Fraaije, M. Monooxygenases as biocatalysts: Classification, mechanistic aspects and biotechnological applications. J. Biotechnol., 2010, 146, 9-24.
[9]
Mohammad, J.; Maghami, S.; Mohammad, B.G.; Mohkam, M.; Zamani, M.; Ebrahimi, N.; Ghasemi, Y. Molecular cloning, characterization and bioinformatics analysis of CYP152A1 gene from Bacillus subtilis in Escherichia coli. Minerva Biotecnol., 2016, 28, 12-18.
[10]
Whitehouse, C.J.; Bell, S.G.; Wong, L.L. P450 BM3 (CYP102A1): Connecting the dots. Chem. Soc. Rev., 2012, 41, 1218-1260.
[11]
Hollmann, F.; Hofstetter, K.; Schmid, A. Non-enzymatic regeneration of nicotinamide and flavin cofactors for monooxygenase catalysis. Trends Biotechnol., 2006, 24, 163-171.
[12]
Nordblom, G.D.; White, R.E.; Coon, M.J. Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450. Arch. Biochem. Biophys., 1976, 175, 524-533.
[13]
Hrycay, E.G.; Gustafsson, J.Å.; Ingelman-Sundberg, M.; Ernster, L. Sodium periodate, sodium chlorite, and organic hydroperoxides as hydroxylating agents in hepatic microsomal steroid hydroxylation reactions catalyzed by cytochrome P-450. FEBS Lett., 1975, 56, 161-165.
[14]
Shoji, O.; Fujishiro, T.; Nagano, S.; Tanaka, S.; Hirose, T.; Shiro, Y.; Watanabe, Y. Understanding substrate misrecognition of hydrogen peroxide dependent cytochrome P450 from Bacillus subtilis. J. Biol. Inorg. Chem., 2010, 15, 1331-1339.
[15]
Budde, M.; Maurer, S.C.; Schmid, R.D.; Urlacher, V.B. Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis. Appl. Microbiol. Biotechnol., 2004, 66, 180-186.
[16]
Cryle, M.J.; Stok, J.E.; De Voss, J.J. Reactions catalyzed by bacterial cytochromes P450. Aust. J. Chem., 2003, 56, 749-762.
[17]
Dix, D.B.; Thompson, R.C. Codon choice and gene expression: Synonymous codons differ in translational accuracy. Proc. Natl. Acad. Sci., 1989, 86, 6888-6892.
[18]
Chen, D.; Texada, D.E. Low-usage codons and rare codons of Escherichia coli. Gene Ther. Mol. Biol., 2006, 10, 1-12.
[19]
Gustafsson, C.; Govindarajan, S.; Minshull, J. Codon bias and heterologous protein expression. Trends Biotechnol., 2004, 22, 346-353.
[20]
Kane, J.F. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol., 1995, 6, 494-500.
[21]
Muhlrad, D.; Parker, R. Premature translational termination triggers mRNA decapping. Nature, 1994, 370, 578.
[22]
Buhr, F.; Jha, S.; Thommen, M.; Mittelstaet, J.; Kutz, F.; Schwalbe, H.; Rodnina, M.V.; Komar, A.A. Synonymous codons direct cotranslational folding toward different protein conformations. Mol. Cell, 2016, 61, 341-351.
[23]
Widmann, M.; Clairo, M.; Dippon, J.; Pleiss, J. Analysis of the distribution of functionally relevant rare codons. BMC Genomics, 2008, 9, 207.
[24]
Chartier, M.; Gaudreault, F.; Najmanovich, R. Large-scale analysis of conserved rare codon clusters suggests an involvement in co-translational molecular recognition events. Bioinformatics, 2012, 28, 1438-1445.
[25]
Gustafsson, C.; Minshull, J.; Govindarajan, S.; Ness, J.; Villalobos, A.; Welch, M. Engineering genes for predictable protein expression. Protein Expr. Purif., 2012, 83, 37-46.
[26]
Burgess-Brown, N.A.; Sharma, S.; Sobott, F.; Loenarz, C.; Oppermann, U.; Gileadi, O. Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study. Protein Expr. Purif., 2008, 59, 94-102.
[27]
Del Tito, B.; Ward, J.M.; Hodgson, J.; Gershater, C.; Edwards, H.; Wysocki, L.A.; Watson, F.A.; Sathe, G.; Kane, J.F. Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli. J. Bacteriol., 1995, 177, 7086-7091.
[28]
Zdanovsky, A.G.; Zdanovskaia, M.V. Simple and efficient method for heterologous expression of clostridial proteins. Appl. Environ. Microbiol., 2000, 66, 3166-3173.
[29]
Goodluck, U. ATGme: Open-source web application for rare codon identification and custom DNA sequence optimization. BMC Bioinformatics, 2015, 16, 303.
[30]
Theodosiou, A.; Promponas, V.J. LaTcOm: A web server for visualizing rare codon clusters in coding sequences. Bioinformatics, 2012, 28, 591-592.
[31]
Thanaraj, T.; Argos, P. Protein secondary structural types are differentially coded on messenger RNA. Protein Sci., 1996, 5, 1973-1983.
[32]
Guex, N.; Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis, 1997, 18, 2714-2723.
[33]
Zhang, Y. I-Tasser server for protein 3D structure prediction. BMC Bioinformatics, 2008, 9, 40.
[34]
Kaplan, W.; Littlejohn, T.G. Swiss-PDB viewer (deep view). Brief. Bioinform., 2001, 2, 195-197.
[35]
DeLano, W.L. Pymol: An open-source molecular graphics tool. CCP4 Newslett. Protein Crystallogr., 2002, 40, 82-92.
[36]
Trott, O.; Olson, A.J. AutoDock vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem., 2010, 31, 455-461.
[37]
Wu, S.; Zhang, Y. LOMETS: A local meta-threading-server for protein structure prediction. Nucleic Acids Res., 2007, 35, 3375-3382.
[38]
Guex, N.; Peitsch, M. Swiss-PdbViewer: A fast and easy-to-use PDB viewer for Macintosh and PC; Protein Data Bank Quat. Newslett, 1996, p. 77.
[39]
Lee, D.S.; Yamada, A.; Sugimoto, H.; Matsunaga, I.; Ogura, H.; Ichihara, K.; Adachi, S.; Park, S.Y.; Shiro, Y. Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis crystallographic, spectroscopic, and mutational studies. J. Biol. Chem., 2003, 278, 9761-9767.
[40]
Vriend, G. WHAT IF: A molecular modeling and drug design program. J. Mol. Graph., 1990, 8, 52-56.
[41]
Tina, K.; Bhadra, R.; Srinivasan, N. Nucleic Acids Res. 35. Web Server issue), 2007, W473-W476
[42]
Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and autodocktools4: Automated docking with selective receptor flexibility. J. Comput. Chem., 2009, 30, 2785-2791.
[43]
OLBoyle. N.M.; Banck, M.; James, C.A.; Morley, C.; Vandermeersch, T.; Hutchison, G.R. Open babel: An open chemical toolbox. J. Cheminform., 2011, 3, 33.
[44]
Dong, H.; Nilsson, L.; Kurland, C.G. Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J. Mol. Biol., 1996, 260, 649-663.
[45]
Varenne, S.; Baty, D.; Verheij, H.; Shire, D.; Lazdunski, C. The maximum rate of gene expression is dependent in the downstream context of unfavourable codons. Biochimie, 1989, 71, 1221-1229.
[46]
Clarke IV, T.F.; Clark, P.L. Rare codons cluster. PLoS One, 2008, 3e3412
[47]
Wallace, A.C.; Laskowski, R.A.; Thornton, J.M. LIGPLOT: A Program to generate schematic diagrams of protein-ligand interactions. Protein Eng., 1995, 8, 127-134.
[48]
Zanger, U.M.; Schwab, M. Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol. Ther., 2013, 138, 103-141.
[49]
Sørensen, M.A.; Kurland, C.; Pedersen, S. Codon usage determines translation rate in Escherichia coli. J. Mol. Biol., 1989, 207, 365-377.
[50]
Varenne, S.; Buc, J.; Lloubes, R.; Lazdunski, C. Translation is a non-uniform process: Effect of tRNA availability on the rate of elongation of nascent polypeptide chains. J. Mol. Biol., 1984, 180, 549-576.
[51]
Zalucki, Y.M.; Jennings, M.P. Experimental confirmation of a key role for non-optimal codons in protein export. Biochem. Biophys. Res. Commun., 2007, 355, 143-148.
[52]
Seidelt, B.; Innis, C.A.; Wilson, D.N.; Gartmann, M.; Armache, J-P.; Villa, E.; Trabuco, L.G.; Becker, T.; Mielke, T.; Schulten, K. Structural insight into nascent polypeptide chain-mediated translational stalling. Science, 2009, 326, 1412-1415.
[53]
Lu, J.; Deutsch, C. Electrostatics in the ribosomal tunnel modulate chain elongation rates. J. Mol. Biol., 2008, 384, 73-86.
[54]
Makrides, S.C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev., 1996, 60, 512-538.
[55]
Shahbazi, M.; Haghkhah, M.; Rahbar, M.R.; Nezafat, N.; Ghasemi, Y. In Silico sub-unit hexavalent peptide vaccine against a Staphylococcus aureus biofilm-related infection. Int. J. Peptide Res. Therapeutics, 2015, 1-17.
[56]
Zamani, M.; Nezafat, N.; Negahdaripour, M.; Dabbagh, F.; Ghasemi, Y. In silico evaluation of different signal peptides for the secretory production of human growth hormone in E. coli. Int. J. Peptide Res. Therapeut., 2015, pp. 1-8.
[57]
Mortazavi, M.; Hosseinkhani, S. Surface charge modification increases firefly luciferase rigidity without alteration in bioluminescence spectra. Enzyme Microb. Technol., 2017, 96, 47-59.
[58]
Mortazavi, M.; Nezafat, N.; Negahdaripour, M.; Gholami, A.; Torkzadeh-Mahani, M.; Lotfi, S.; Ghasemi, Y. In silico evaluation of rare codons and their positions in the structure of cytosine deaminase and substrate docking studies. Trends Pharmacol. Sci., 2016, 2, 117-130.
[59]
Bina, S.; Shenavar, F.; Khodadad, M.; Haghshenas, M.; Mortazavi, M.; Fattahi, M.; Erfani, N.; Hosseini, S. Impact of RGD peptide tethering to IL24/mda-7 (melanoma differentiation associated gene-7) on apoptosis induction in hepatocellular carcinoma cells. Asian Pac. J. Cancer Prev., 2014, 16, 6073-6080.
[60]
Mortazavi, M.; Hosseinkhani, S. Design of thermostable luciferases through arginine saturation in solvent-exposed loops. Protein Engr. Des. Select., 2011, gzr051.
[61]
Kargar, F.; Mortazavi, M.; Savardashtaki, A.; Hosseinkhani, S.; Mahani, M.T.; Ghasemi, Y. Genomic and protein structure analysis of the luciferase from the Iranian bioluminescent beetle, Luciola sp. Int. J. Biol. Macromol., 2019, 124, 689-698.
[62]
Yousefi, F.; Ataei, F.; Mortazavi, M.; Hosseinkhani, S. Bifunctional role of leucine 300 of firefly luciferase in structural rigidity. Int. J. Biol. Macromol., 2017, 101, 67-74.
[63]
Fattahi, M.; Malekpour, A.; Mortazavi, M.; Safarpour, A.; Naseri, N. The characteristics of rare codon clusters in the genome and proteins of hepatitis C virus; a bioinformatics look. Middle East J. Dig. Dis., 2014, 6, 214.
[64]
Mortazavi, M.; Zarenezhad, M.; Gholamzadeh, S.; Alavian, S.M.; Ghorbani, M.; Dehghani, R.; Malekpour, A.; Meshkibaf, M.; Fakhrzad, A. Bioinformatics identification of rare codon clusters (RCCs) in HBV genome and evaluation of RCCs in HBV proteins structure of hepatitis B virus. Hepatitis Monthly, 2016, 16(10)e39909
[65]
Mortazavi, M.; Zarenezhad, M.; Alavian, S.M.; Gholamzadeh, S.; Malekpour, A.; Ghorbani, M. TorkzadehMahani, M.; Lotfi, S.; Fakhrzad, A. Bioinformatic analysis of codon usage and phylogenetic relationships in different genotypes of the hepatitis C virus. Hepatitis Monthly, 2016, 16(10)e39196
[66]
Rodrigues, J.; Araújo, R.; Prather, K.L.; Kluskens, L.; Rodrigues, L. Heterologous production of caffeic acid from tyrosine in Escherichia coli. Enzyme Microb. Technol., 2015, 71, 36-44.
[67]
Stahlhut, S.G.; Siedler, S.; Malla, S.; Harrison, S.J.; Maury, J.; Neves, A.R.; Forster, J. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli. Metab. Eng., 2015, 31, 84-93.
[68]
Guengerich, F.P. Cytochrome P450; Springer, 1995, p. 473-535.
[69]
Ogura, H.; Nishida, C.R.; Hoch, U.R.; Perera, R.; Dawson, J.H.; Ortiz de Montellano, P.R. EpoK, a cytochrome P450 involved in biosynthesis of the anticancer agents epothilones A and B. Substrate-mediated rescue of a P450 enzyme. Biochemistry, 2004, 43, 14712-14721.
[70]
Denisov, I.G.; Makris, T.M.; Sligar, S.G.; Schlichting, I. Structure and chemistry of cytochrome P450. Chem. Rev., 2005, 105, 2253-2278.


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VOLUME: 17
ISSUE: 1
Year: 2020
Published on: 06 January, 2020
Page: [10 - 22]
Pages: 13
DOI: 10.2174/1570164616666190220143131
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