Category: Assay Development and Screening
We report here the substrate specificity and kinetic mechanism of the bifunctional N-acetyltransferase, uridyltransferase, GlmU, from M. tuberculosis (MtGlmU). This enzyme is responsible for the final two steps of the synthesis of UDP-N-acetylglucosamine, which is an essential precursor of peptidoglycan. In order to catalyze the transfer of an acetyl group from acetyl-coenzyme A (AcCoA) to glucosamine 1-phosphate (GlcN-1P) to form N-acetylglucosmaine-1-phosphate, we have found that MtGlmU forms a ternary complex with the aforementioned substrates prior to catalysis. Product and dead-end inhibition studies in combination with differential scanning fluorimetry binding studies were used to define the order of substrate binding and product release. pH dependence of kcat and kcat/Km studies demonstrate that MtGlmU employs two general bases and two general acids for maximal activity (the general bases have pKa values of ca. 6.5 and 7.5, while the general acids have pKa values of ca. 8.3 and 9.1). In addition we sought to determine the protonation profile of glucosamine 1-phosphate, using 1H-NMR spectroscopy.
Additionally, we have carried out kinetic, small molecule screening studies in order to identify inhibitors of the MtGlmU acetyltransferase domain and here we present this initial data along with some early biophysical studies to confirm the inhibition of identified compounds prior to further experiments.
Peter Craggs– Investigator, The Francis Crick Institute, UCL, London, England, United Kingdom