Insights into Pyrazinamidase and DNA Gyrase Protein Structures in Resistant and Susceptible Clinical Isolates of Mycobacterium tuberculosis.

BACKGROUND: Mutations in pncA and gyrA genes cause pyrazinamide (PZA) and fluroquinolone resistance in Mycobacterium tuberculosis (MTB). In the present study, structures of pyrazinamidase (PZase) and DNA gyrase proteins were studied in resistant and susceptible clinical isolates of MTB. MATERIALS AND METHODS: Sixty clinical isolates of MTB were used in this study. Polymerase chain reaction (PCR) amplification of pncA and gyrA genes was accomplished on purified DNA. Sequence of the fragments was determined by an Applied BiosystemsTM apparatus. Bioinformatic analysis was performed by online software and three-dimensional (3D) structures of proteins was predicted using Molegro Virtual Docker (MVD) Modeler software. RESULTS: Amplified 744 and 194 bp fragments of pncA and gyrA genes, respectively were yielded suitable sequence results. Predicted 3D structures of proteins showed some differences between wild-type and mutant structures. Mutation in amino acid No.31 (T92C) caused an increase in distance from metal ion position to enzyme active site, but it was considered as a polymorphism. Docking results by MVD revealed a relationship in quinolone resistance-determining regions (QRDR) amino acids in interaction with antibiotic. T92C mutation in PZase from non-polar aliphatic amino acid Ile (ATC) to polar aliphatic amino acid threonine (ACC) was a polymorphism. CONCLUSION: Structural changes in two important proteins related to drug resistance were proven in clinical isolates of MTB.

A novel biological role for nsLTP2 from Oriza sativa: Potential incorporation with anticancer agents, nucleosides and their analogues.

Development of a protein-based drug delivery system has major impact on the efficacy and bioavailability of unstable and water insoluble drugs. In the present study, the binding modes of a nonspecific lipid transfer protein (nsLTP2) from Oryza sativa with various nucleosides and analogous molecules were identified. The 3-D structure of the protein was designed and validated using modeler 9.13, Molegro virtual docker and procheck tool, respectively. The binding affinity and strength of interactions, key contributing residues and specificity toward the substrates were accomplished by computational docking and model prediction. The protein presented high affinity to acyclovir and vidarabine as purine-analogous drugs. Binding affinity is influenced by the core template and functional groups of the ligands which are structurally different cause the variation of interaction energies with nsLTP2. Nonetheless, all the evaluated analogous drugs occupy the proximity space at the nsLTP active site with high similarity in their binding modes. Our findings hold great promise for the future applications of nsLTPs in various aspects of pharmaceutical science and molecular biology.

Computational evaluation on the binding affinity of non-specific lipid-transfer protein-2 with fatty acids.

A computational study was carried out to identify the structural determinant controlling the affinity, specificity and binding strength of several saturated and unsaturated fatty acids with Oryza sativa (Indica group) nonspecific lipid transfer protein (nsLTP2). Association between the number, position and conformation of hydrophobic patches and lipid binding properties of the protein was evidenced by docking analysis. Binding affinity is influenced by the number of carbon atoms, location of double bonds and hydroxyl group in the acyl chain. The results may direct at developing applications in LTP-mediated transport and controlled release of low molecular weight drugs.

Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis.

INTRODUCTION: Pyrazinamide is one of the first line four drugs for treatment of tuberculosis. It was proved that mutations in two nucleotides of 359 and 374 pnc genes are highly associated with resistance to pyrazinamide. MATERIALS AND METHODS: In this study, mutations in these two codones in 30 clinical isolates of Mycobacterium tuberculosis were detected by means of sequencing. Protein structures encoded by this gene with and without mutation were investigated in resistant and susceptible isolates to pyrazinamide, respectively. RESULTS: Mutation in the positions 359 and 374 altered some parameters like change in electronic charge, distance change of mutated amino acids to situation of active enzyme and metal connection situation. In these conditions, structure and function of pyrozinamidase enzyme were changed and antibiotic was ineffective and consequently caused resistance to pyrazinamide in M. tuberculosis. CONCLUSION: This work was revealed protein changes in resistance to pyrazinamide in clinical isolates of M. tuberculosis.