Vitamin B1 is an indispensable co-factor for various enzymes inter alia in the Krebs cycle, pentose phosphate pathway, nucleotide and amino acid synthesis. Due to its importance in metabolism, proteins involved in the synthesis of vitamin B1 have been identified as potential drug targets. Thiamine monophosphate kinase (ThiL), catalyses the last reaction in the pathway, the ATP dependent phosphorylation of thiamine monophosphate (TMP) producing thiamine pyrophosphate (TPP) the active and co-factor form of vitamin B1.
In this study, thiamine monophosphate kinase from Mycobacterium tuberculosis (MtbThiL, ~36 kDa) was produced as an N-terminally His6-tagged fusion protein, purified by affinity and size exclusion chromatography, and crystallised. Hexagonal MtbThiL crystals belonged to space group P6122. Molecular replacement revealed a symmetric homodimer with a single monomer occupying the asymmetric unit. Analysis of the structure showed that each subunit of MtbThiL has an ATP and TMP binding site and is structurally related to other ThiL enzymes.
Ten lead compounds were identified from compound databases as potential ThiL inhibitors, and oxythiamine was chosen for further study. The binding affinities of oxythiamine and TMP to MtbThiL were determined by isothermal titration calorimetry and a pyruvate kinase-lactate dehydrogenase enzyme assay, which revealed that the binding affinity for oxythiamine by MtbThiL is lower than the substrate TMP. / Dissertation (MSc)--University of Pretoria, 2020. / Biochemistry / MSc / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/77810 |
| Date | January 2020 |
| Creators | Dlamini, Lenye Sebenzile |
| Contributors | Schubert, Wolf-Dieter, u12140164@tuks.co.za |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2020 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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