Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough ...understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in V
for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.
Anyone in England over 18 whose health or wellbeing suddenly deteriorates at home will have access to an urgent community response (UCR) team within 2 hours by April 2022. Community nursing services ...are providing the core service model to provide these crisis response services. Nurse leads from three UCR accelerator sites (Kirklees, Warrington and Bromley) elaborate on how they are making waves of change for the better, using their clinical skills and building key relationships with other health services. Acutely unwell patients are being seen by practitioners with advanced assessment skills, which keeps eight out of 10 patients at home safe, avoiding hospital admission.
The capacity for some pathogens to jump into different host-species populations is a major threat to public health and food security. Staphylococcus aureus is a multi-host bacterial pathogen ...responsible for important human and livestock diseases. Here, using a population-genomic approach, we identify humans as a major hub for ancient and recent S. aureus host-switching events linked to the emergence of endemic livestock strains, and cows as the main animal reservoir for the emergence of human epidemic clones. Such host-species transitions are associated with horizontal acquisition of genetic elements from host-specific gene pools conferring traits required for survival in the new host-niche. Importantly, genes associated with antimicrobial resistance are unevenly distributed among human and animal hosts, reflecting distinct antibiotic usage practices in medicine and agriculture. In addition to gene acquisition, genetic diversification has occurred in pathways associated with nutrient acquisition, implying metabolic remodelling after a host switch in response to distinct nutrient availability. For example, S. aureus from dairy cattle exhibit enhanced utilization of lactose-a major source of carbohydrate in bovine milk. Overall, our findings highlight the influence of human activities on the multi-host ecology of a major bacterial pathogen, underpinned by horizontal gene transfer and core genome diversification.
Antimicrobial resistance represents a growing global health problem. The emergence of novel resistance mechanisms necessitates the development of alternative approaches to investigate the molecular ...fundamentals of resistance, leading ultimately to new strategies for counteracting them. To gain deeper insight into antibiotic-target interactions, the binding of the frontline anti-tuberculosis drug isoniazid (INH) to a target enzyme, InhA, from
Mycobacterium tuberculosis
was studied using ultrafast two-dimensional infrared (2D-IR) spectroscopy and molecular simulations. Comparing wild-type InhA with a series of single point mutations, it was found that binding of the INH-NAD inhibitor to susceptible forms of the enzyme increased the vibrational coupling between residues located in the Rossmann fold co-factor binding site of InhA and suppressed dynamic fluctuations of the enzyme structure. The effect correlated with biochemical assay data, being reduced in the INH-resistant S94A mutant and absent in the biochemically-inactive P193A control. Molecular dynamics simulations and calculations of inter-residue couplings indicate that the changes in coupling and dynamics are not localised to the co-factor binding site, but permeate much of the protein. We thus propose that the resistant S94A mutation circumvents subtle changes in global structural dynamics caused by INH upon binding to the wild-type enzyme that may impact upon the formation of important protein-protein complexes in the fatty acid synthase pathway of
M. tuberculosis
.
2D-IR spectroscopy reveals a role for protein structural dynamics in antimicrobial-resistance.
2D-IR spectroscopy reveals a role for protein structural dynamics in antimicrobial-resistance.
Antimicrobial resistance represents a growing global health problem. The emergence of novel resistance ...mechanisms necessitates the development of alternative approaches to investigate the molecular fundamentals of resistance, leading ultimately to new strategies for counteracting them. To gain deeper insight into antibiotic–target interactions, the binding of the frontline anti-tuberculosis drug isoniazid (INH) to a target enzyme, InhA, from
Mycobacterium tuberculosis
was studied using ultrafast two-dimensional infrared (2D-IR) spectroscopy and molecular simulations. Comparing wild-type InhA with a series of single point mutations, it was found that binding of the INH–NAD inhibitor to susceptible forms of the enzyme increased the vibrational coupling between residues located in the Rossmann fold co-factor binding site of InhA and suppressed dynamic fluctuations of the enzyme structure. The effect correlated with biochemical assay data, being reduced in the INH-resistant S94A mutant and absent in the biochemically-inactive P193A control. Molecular dynamics simulations and calculations of inter–residue couplings indicate that the changes in coupling and dynamics are not localised to the co-factor binding site, but permeate much of the protein. We thus propose that the resistant S94A mutation circumvents subtle changes in global structural dynamics caused by INH upon binding to the wild-type enzyme that may impact upon the formation of important protein–protein complexes in the fatty acid synthase pathway of
M. tuberculosis
.
The results of infrared spectroscopic investigations into the band assignments, vibrational relaxation, and solvation dynamics of the common anti-tuberculosis treatment Isoniazid (INH) are reported. ...INH is known to inhibit InhA, a 2-trans-enoyl-acyl carrier protein reductase enzyme responsible for the maintenance of cell walls in Mycobacterium tuberculosis but as new drug-resistant strains of the bacterium appear, next-generation therapeutics will be essential to combat the rise of the disease. Small molecules such as INH offer the potential for use as a biomolecular marker through which ultrafast multidimensional spectroscopies can probe drug binding and so inform design strategies but a complete characterization of the spectroscopy and dynamics of INH in solution is required to inform such activity. Infrared absorption spectroscopy, in combination with density functional theory calculations, is used to assign the vibrational modes of INH in the 1400-1700 cm(-1) region of the infrared spectrum while ultrafast multidimensional spectroscopy measurements determine the vibrational relaxation dynamics and the effects of solvation via spectral diffusion of the carbonyl stretching vibrational mode. These results are discussed in the context of previous linear spectroscopy studies on solid-phase INH and its usefulness as a biomolecular probe.
Determining the chemical and structural modifications occurring within a protein during fundamental processes such as ligand or substrate binding is essential to building up a complete picture of ...biological function. Currently, significant unanswered questions relate to the way in which protein structural dynamics fit within the structure-function relationship and to the functional role, if any, of bound water molecules in the active site. Addressing these questions requires a multidisciplinary approach and complementary experimental techniques that, in combination, enhance our understanding of the complexities of protein chemistry. We exemplify this philosophy by applying both physical and biological approaches to investigate the active site chemistry that contributes to the inhibition of the Corynebacterium glutamicum catalase enzyme by nitric oxide. Ultrafast two-dimensional infrared spectroscopy (2D-IR) experiments exploit the NO ligand as a local probe of the active site molecular environment and shows that catalase displays a dynamically-restricted, 'tight,' structure. X-ray crystallography studies of C. glutamicum catalase confirm the presence of a conserved chain of hydrogen-bonded bound water molecules that link the NO ligand and the protein scaffold. This combination of bound water and restricted dynamics stands in stark contrast to other haem proteins, such as myoglobin, that exhibit ligand transport functionality despite the presence of a similar distal architecture in close proximity to the ligand. We conclude not only that the bound water molecules in the catalase active site play an important role in molecular recognition of NO but also may be part of the mechanistic operation of this important enzyme.
The ultrafast equilibrium fluctuations of the Fe(III)-NO complex of a single point mutation of Myoglobin (H64Q) have been studied using Fourier Transform 2D-IR spectroscopy. Comparison with data from ...wild type Myoglobin (wt-Mb) shows the presence of two conformational substates of the mutant haem pocket where only one exists in the wild type form. One of the substates of the mutant exhibits an almost identical NO stretching frequency and spectral diffusion dynamics to wt-Mb while the other is distinctly different in both respects. The remarkably contrasting dynamics are largely attributable to interactions between the NO ligand and a nearby distal side chain which provides a basis for understanding the roles of these side chains in other ferric haem proteins.
The GntR super-family of transcriptional regulators are ubiquitously distributed throughout the prokaryotic world. There are currently 231,015 sequences in the Pfam database pertaining to GntR ...proteins (PF000392) from over 17,000 bacterial species. The proteobacteria, firmicutes and actinobacetria account for 95% of all GntR sequences in the database. Yet despite their wide distribution, very few have been studied in depth. The general structure of GntR proteins is a highly homologous helix-turn-helix (HTH) domain linked to a C-terminal effector binding/oligomerisation (Eb/O domain) by a flexible linker. Binding of effector molecules to the Eb/O domain causes conformational changes within the protein allowing binding or release of specific DNA operator sequences which controls gene transcription or repression. The work described here aims to address some of the unknowns relating to GntR-like regulators in terms of structural and dynamical information about their general mechanism of function of this highly diverse family of proteins. Thirty target GntR proteins were selected from sequence analysis of PF000392. These thirty targets were extensively analysed in silico revealing four proteins with novel C-terminal topologies (Gp26, FucR, Reut_B4779 and Colbol_00895) for which there is no structural information. Four proteins (HutC, DevA, DevE and Gp26) were also studied experimentally by using protein biochemistry and x-ray crystallography. The structure of HutC has been solved. However, it has not been fully refined due to the publication of a homologous structure during the course of this work. DevE crystals were obtained and examined by x-ray, further refinement of crystallography conditions and selenomethionine protein preparation resulted in poor phasing and low resolution defracting crystals. Work in this thesis also aimed to refine methodology of 2D-IR spectroscopy to examine protein molecular dynamics at femtosecond resolution with a view to applying this to GntR-like proteins. The model system for this work was InhA and isoniazid.
The GntR super-family of transcriptional regulators are ubiquitously distributed throughout the prokaryotic world. There are currently 231,015 sequences in the Pfam database pertaining to GntR ...proteins (PF000392) from over 17,000 bacterial species. The proteobacteria, firmicutes and actinobacetria account for 95% of all GntR sequences in the database. Yet despite their wide distribution, very few have been studied in depth. The general structure of GntR proteins is a highly homologous helix-turn-helix (HTH) domain linked to a C-terminal effector binding/oligomerisation (Eb/O domain) by a flexible linker. Binding of effector molecules to the Eb/O domain causes conformational changes within the protein allowing binding or release of specific DNA operator sequences which controls gene transcription or repression. The work described here aims to address some of the unknowns relating to GntR-like regulators in terms of structural and dynamical information about their general mechanism of function of this highly diverse family of proteins. Thirty target GntR proteins were selected from sequence analysis of PF000392. These thirty targets were extensively analysed in silico revealing four proteins with novel C-terminal topologies (Gp26, FucR, Reut_B4779 and Colbol_00895) for which there is no structural information. Four proteins (HutC, DevA, DevE and Gp26) were also studied experimentally by using protein biochemistry and x-ray crystallography. The structure of HutC has been solved. However, it has not been fully refined due to the publication of a homologous structure during the course of this work. DevE crystals were obtained and examined by x-ray, further refinement of crystallography conditions and selenomethionine protein preparation resulted in poor phasing and low resolution defracting crystals. Work in this thesis also aimed to refine methodology of 2D-IR spectroscopy to examine protein molecular dynamics at femtosecond resolution with a view to applying this to GntR-like proteins. The model system for this work was InhA and isoniazid.
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