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: Pathogenic microorganisms -- Analysis , Drug resistance in microorganisms , Microorganisms -- Effect of drugs on , Antibiotics -- Effectiveness , Pathogenic bacteria , Drug tolerance , Enzymes -- Analysis , Peptide antibiotics
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/161911 , vital:40690
- 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: Pathogenic microorganisms -- Analysis , Drug resistance in microorganisms , Microorganisms -- Effect of drugs on , Antibiotics -- Effectiveness , Pathogenic bacteria , Drug tolerance , Enzymes -- Analysis , Peptide antibiotics
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/161911 , vital:40690
- 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
The investigation of novel marine microorganisms for the production of biologically active metabolites
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
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