Sintra Pisco, João;
(2018)
Studies on the mechanism of allosteric regulation of M. tuberculosis ATP-phosphoribosyltransferase.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Allosteric regulation is an efficient way of controlling enzymatic activity. In Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, ATPphosphoribosyltransferase (ATP-PRT) catalyses the first and committed step of the biosynthesis of L-histidine (L-His). L-His biosynthetic pathway is essential for Mtb and is absent in humans, making ATP-PRT an attractive target for the development of novel antibiotics. ATP-PRT is a hexamer in solution and, like other enzymes regulated via ferrodoxin-like (FL) domains, interconverts between an open active and a closed inactive conformation. Binding of L-His, its feedback allosteric inhibitor, shifts the equilibrium towards the closed state. The exact mechanisms of allosteric control of ATP-PRT activity are currently poorly understood. Likewise, it is unknown if perturbations in the allosteric regulation of ATP-PRT would be detrimental to Mtb. In this work, I characterised the allosteric regulation of ATP-PRT by L-His employing enzymology, biophysics, native mass spectrometry and X-ray crystallography. Using analogues of L-His, I showed that the allosteric inhibition is specific for L-His. A new targeted compound screen for possible allosteric regulators was developed and led to the discovery of 3-(2-Thienyl)-L-alanine (TIH) as an allosteric activator. Kinetic and structural analyses revealed uncoupling between ATP-PRT activity and conformational changes. In order to understand the molecular basis of allosteric regulation of ATP-PRT by L-His, I generated and analysed ATP-PRT variants with point mutations in the allosteric site. These mutations had a direct effect on ATP-PRT stability, activity and allosteric regulation by L-His and TIH. Overall, the results of this work show a complex allosteric regulation of ATPPRT by L-His while revealing the possibility of allosteric activation. Results with the ATP-PRT variants will guide us towards a detailed characterization of the allosteric control of ATP-PRT and its importance for Mtb, which might have implications to other FL domain-containing enzymes and prove useful for drug discovery.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Studies on the mechanism of allosteric regulation of M. tuberculosis ATP-phosphoribosyltransferase |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10046004 |
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