Analysis of bacterial Mur amide ligase enzymes for the identification of inhibitory compounds by in silico methods
- Chamboko, Chiratidzo Respina
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
- Authors: Chamboko, Chiratidzo Respina
- Date: 2020
- Subjects: Mur amide ligases , Ligases , Ligand binding (Biochemistry) , Antibacterial agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163430 , vital:41036
- Description: An increased emergence of resistant pathogenic bacterial strains over the years has resulted in many people dying of untreatable infections. This has become one of the most critical global public health problems, as resistant strains are complicating treatment of infectious diseases, increasing human morbidity, mortality, and health care costs. A very limited amount of effective antibiotics is currently available, but the development of novel classes of antibacterial agents is becoming a priority. Mur amide ligases are enzymes that have been identified as potentially good targets for antibiotics, as they are uniquely found in bacteria. They are responsible for the formation of peptide bonds in a growing peptidoglycan structure for bacterial cell walls. The current work presented here focused on characterizing these Mur amide ligase enzymes and obtaining inhibitory compounds that could potentially be of use in drug discovery of antibacterial agents. To do this, multiple sequence alignment, motif analysis and phylogenetic tree constructions were carried out, followed by docking studies and molecular dynamic simulations. Prior to docking, homology modelling of missing residues in the MurF structure (PDB 1GG4) was performed. Characterization results revealed the Mur amide ligase enzymes contained defined conservation in limited regions, that ultimately mapped towards the central domain responsible for ATP binding (presence of a conserved GKT motif). Further analysis of results further unraveled the unique patterns observed within each group of the family of enzymes. As a result of these findings, docking studies were carried out on each Mur amide ligase structure. At most, two ligands were identified to be sufficiently inhibiting each Mur amide ligase. The ligands obtained were SANC00574 and SANC00575 for MurC, SANC00290 and SANC00438 for MurD, SANC00290 and SANC00525 for MurE and SANC00290 and SANC00434 for MurF. The two best ligands identified for each enzyme had docked in the active site of their respective proteins, passed Lipinski’s rule of five and had substantially low binding energies. Molecular dynamic simulations were then performed to analyze the behavior of the proteins and protein-ligand complexes, to confirm the lead compounds as good inhibitors of the Mur amide ligases. In the case of MurC, MurD and MurE complexes, the identified ligands clearly impacted the behavior of the protein, as the ligand bound proteins became more compact and stable, while flexibility decreased. There was however an opposite effect on MurF complexes, that resulted in identified inhibitors being discarded. As a potential next step, in vivo and in vitro experiments can be performed with identified ligands from this research, to further support the information presented.
- Full Text:
- Date Issued: 2020
Sequence and structural investigation of the nonribosomal peptide synthetases of Bacillus atrophaeus UCMB 5137(63Z)
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
- Authors: Ryan, Candice Nancy
- Date: 2013 , 2013-04-19
- Subjects: Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3891 , http://hdl.handle.net/10962/d1003057 , Bacillus (Bacteria) , Peptides--Synthesis , Antibiotics , Drug resistance in microorganisms , Amino acids , Phytopathogenic microorganisms , Trees--Phylogeny , Ligases
- Description: Due to increased plant resistance to the existing antibiotics produced, there is a need to develop alternatives. Nonribosomal peptides (NRPs) are important plant phytopathogens synthesized by nonribosomal peptide synthetases (NRPSs). In this study, a newly sequenced Bacillus strain Bacillus atrophaeus UCMB 5137 (63Z), found to have increased phytopathogenic activity, was investigated to gain insights to the possible reason behind this activity. NRPS modules were identified using a novel script that can act on unannotated, raw DNA sequences. The Structure Based Sequence Analysis Webserver was used to identify the amino acids incorporated into the final NRP, which were compared to the NRP database. Five NRPSs were found within the strain; fengycin/plipstatin, mycosubtilin, surfactin, bacillibactin and bacitracin. Some of the modules usually present for these NRPSs were not present in the test strain and only a few modules were found. A phylogenetic study was carried out and the topologies of the trees showed that genes were not transferred horizontally. It did, however, lead to the hypothesis that different NRPS genes are under different adaptive evolutionary pressures. Only slight conformational changes between L and D-conformation of amino acids were seen between the test and neighboring strains. All of the linker and terminal regions of synthetases were found to exhibit a large amount of conservation overall. Homology modeling was performed on the test strain on selected modules, TE and A-domains of fengycin and mycosubtilin synthetases. TE-domains between the different synthetases are different and specific for the NRP they facilitate release for. The NRPS from which the A-domain originates also influences substrate specificity as well as the module in which the A-domain occurs within the NRPS. Binding pockets of A-domains of differing substrate specificity were compared. Future work will include; refinement of the models and docking studies within the A-domain binding pocket. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
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