Dataset Information

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs.

ABSTRACT: The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the ?3?3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ?-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two ?-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the ?-helices adopt an "up" state where the ?-helices enter the ?3?3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The ?E-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.

SUBMITTER: Zhang AT

PROVIDER: S-EPMC6410841 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The epidemiological importance of mycobacterial species is indisputable, and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important bacterial metabolites, represents a set of targets for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene disruption, gene knockdown, point mutations (D61W, R134A, E321N), and kinetic analysis to evaluate aroA gene essentiality and vulnerability of its protein product, EPSPS, from Mycolicibacterium (Mycobacterium) smegmatis (MsEPSPS). We demonstrate that aroA-deficient cells are auxotrophic for aromatic amino acids (AroAAs) and that the growth impairment observed for aroA-knockdown cells grown on defined medium can be rescued by AroAA supplementation. We also evaluated the essentiality of selected MsEPSPS residues in bacterial cells grown without AroAA supplementation. We found that the catalytic residues R134 and E321 are essential, while D61, presumably important for protein dynamics and suggested to have an indirect role in catalysis, is not essential under the growth conditions evaluated. We have also determined the catalytic efficiencies (Kcat/Km) of recombinant wild-type (WT) and mutated versions of MsEPSPS (D61W, R134A, E321N). Our results suggest that drug development efforts toward EPSPS inhibition may be ineffective if bacilli have access to external sources of AroAAs in the context of infection, which should be evaluated further. In the absence of AroAA supplementation, aroA from M. smegmatis is essential, its essentiality is dependent on MsEPSPS activity, and MsEPSPS is vulnerable. IMPORTANCE We found that cells from Mycobacterium smegmatis, a model organism safer and easier to study than the disease-causing mycobacterial species, when depleted of an enzyme from the shikimate pathway, are auxotrophic for the three aromatic amino acids (AroAAs) that serve as building blocks of cellular proteins: l-tryptophan, l-phenylalanine, and l-tyrosine. That supplementation with only AroAAs is sufficient to rescue viable cells with the shikimate pathway inactivated was unexpected, since this pathway produces an end product, chorismate, that is the starting compound of essential pathways other than the ones that produce AroAAs. The depleted enzyme, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), catalyzes the sixth step of shikimate pathway. Depletion of this enzyme inside cells was performed by disrupting or silencing the EPSPS-encoding aroA gene. Finally, we evaluated the essentiality of specific residues from EPSPS that are important for its catalytic activity, determined with experiments of enzyme kinetics using recombinant EPSPS mutants.

Dataset Information

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets