Cytochrome P450 monooxygenase analysis in the genus mycobacterium: special focus on CYP123 / by Mohammad Parvez

Abstract

One-third of the human population is infected by latent tuberculosis (TB). Active TB can be cured with drugs unless it is drug-resistant. The study showed that P450 CYP123A1 is highly expressed during the latent phase of M. tuberculosis, the infectious agent that causes TB. Based on meta-analysis of the expression data, CYP123A1 is selected as the best drug candidate against the dormant phase of M. tuberculosis. It is noteworthy that CYP123A1 is present only in TB-causing bacteria, suggesting its essential role. Despite the grate importance of CYP123A1 as drug target for both latent TB and active TB, to date this P450s has not been characterised for its function or for its structure. This study is the first of its kind on M. tuberculosis P450 CYP123 characterisation. Furthermore, the distribution of P450s in the genus Mycobacterium is unknown. Thus, in this study, the molecular evolutionary dynamics of P450s were examined with special focus on mycobacterial P450s. In this study, 17 598 P450s belonging to 113 P450 families (bacteria − 42; fungi − 19; plant − 28; animal − 22; plant and animal − 1 and common P450 family − 1) were analysed and highly conserved and rapidly evolving P450 families was identified. The results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function, such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and This study’s results offer new understanding of the dynamic structural nature of P450s. In silico structural analysis of CYP123A1 is carried out where a 3D homology model of CYP123A1 is constructed using the template 3A4G. The 3D model of CYP123A1 was found to be of high quality, based on validation programmes. The docking of model CYP123A1 with the ligands clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, posaconazole and voriconazole was performed in Autodock vina. The molecular docking studies showed that ketoconazole forms a much better complex than other azole drugs, with the best interaction rate and lowest energy of -9.0 kcal/mol. ketoconazole forms a tight hydrogen bond with residue Lys246 of CYP123A1. Clotrimazole did not show interaction with the 3D model of CYP123A1. Lys246 forms a hydrogen bond with fluconazole and heme forms two Pi-Pi stacking with fluconazole. Voriconazole is involved in one Pi-Cation interaction and hydrogen bond with the CYP123A1 residues Arg82. In order to validate the in silico studies, CYP123A1 was cloned and recombinant cells carrying CYP123A1 in a novel expression vector was generated. In conclusion, the multiple cloning site of the pINK-A vector was successfully modified by adding more suitable restriction enzymes and the revised vector was named pINK-d. Sixteen restriction enzymes were added in such a way that they did not cause any shift or change in the reading frame of the vector. The new vector can be used in future for the cloning of other P450s. CYP123A1 cDNA and the modified expression vector were successfully synthesised by GenScript. Synthesised CYP123A1 was further cloned into expression vector pINK-d. The construct containing the CYP123A1 gene and the empty pINK-d vector were transformed into E. coli cells and recombinant cells were selected on Luria-Bertani ampicillin plates. Plasmids were isolated and the presence of the correct size insert (CYP123A1) was verified by restriction enzyme digestion analysis. Recombinant E. coli cells carrying CYP123A1 and an expression vector were stored at -80oC for future expression and functional analysis of CYP123A1.