Expression, Purification And Kinetics Of M. Tuberculosis GlmU

As a global issue, tuberculosis (TB) is still an infectious disease with extremely high mobidity and mortality, catched by nearly three thirds of worldwide populations, moreover the incidence is rising yearly. Nowadays, due to the continuous increasing of multidrug-resistant tuberculosis (MDR- TB), it turns hard to cure tuberculosis again. Therefore, identifying new targets from M. tuberculosis itself and developing new anti-tuberclosis drugs may become a chief task currently.Certainly, the selection of targets is relevant to the structure of mycobacterial cell wall, which is the chief part for its survival. The core of cell wall has been present as the focus of study for its specific structure, which is composed of peptidoglycan, next to the membranes, arabino- galactan, and mycolic acid. The mycolic acid and arabinogalactan are cov- alently attached to peptidoglycan via the disaccharide linker (L-rhamnosyl- N-acety-l-glucosaminyl-phosphate). UDP-GlcNAc is the active state of GlcNAc and formed through four steps. GlmU, as a bifunctional enzyme, catalyzes transacetylation and transuridylation reactions. The glmU gene knockout model has confirmed its essentiality to mycobacterial survival. The biosynthesis pathway of UDP-GlcNAc is different between bacteria and human and glucosamine-1-P acetyltransferase is absent in human. Therefore, GlmU, espectially its acetyltransferase is a potential drug target. Studies on acetyltransferase of GlmU will be beneficial for developing more safe and reliable anti-tuberclosis drugs.The objectives of this study: (1) to overexpress M. tuberculosis GlmU protein by pET16b express vector in E.coli BL21(DE3). (2) to purify GlmU protein by affinity chromatography and confirm the purified protein by SDS-PAGE and Western blotting. (3) to set up enzyme assays and choose the optimal ones. (4) to study the kinetic of GlmU to understand the optimal catalytic conditions and to detect the catalytic constant Km and Vmax.The results are followings:1. M. tuberculosis GlmU protein was expressed in E.coli BL21(DE3).GlmU protein was induced with 1mM IPTG, 30oC for 3.5 hours. After sonication, both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. The GlmU protein with expected mole- cular mass 54.10 kD is soluble.2. GlmU protein was purified by affinity chromatography.The N-terminus of GlmU was fused with histidine tag in pET16b, therefore GlmU was purified by histidine-Ni2+ affinity chromatography. Elution fractions 1-5 were detected by SDS-PAGE and the purified GlmU protein was present in fractions 2-5. The GlmU protein in elution fractions 3-5 were confirmed by Western blotting. The elution fraction 4 (442μg/ml) was used for further kinetic study.3. The enzyme assays of GlmU were set up.GlmU acetyltransferase activity:(1) HPLC: The product CoA was separated using a reverse-phase column, Nova-Pak C18 (3.9×150 mm, 4μm) at a flow rate of 1 mLmin-1 under phosphate buffer (pH 6.5)-methanol (95:5) and was monitored at 259 nm. The retention time of CoA was 9.6 min, which indicated GlmU has acetyltranferase activity.(2) Medical colorimetric assays: DTNB (Ellman's Reagent) was added into the reaction system to detect the amount of thiols in product CoA. The OD value at 405 nm was read.GlmU uridyltransferase activity:(1) HPLC: UDP-GlcNAc was separated with Nova-Pak C18 at a flow rate of 0.5mLmin-1 under 20 mM triethylamine-acetic acid buffer (pH 4.0) and was monitored at 260 nm. The peak of UDP-GlcNAc was appeared at 7.8 min approximately. It indicated that GlmU has uridyltransferase activity.(2) Medical colorimetric assays: The produced pyrophosphate in the reation was converted into inorganic phosphate by pyrophosphatase. The reaction of inorganic phosphate and ammonium molybdate results in the formation of molybdenum blue, which turns malachite green from yellow to blue. The OD value at 630 nm was detected.4. Kinetic parameters of GlmU protein were determined.(1) Initial velocity was determined.GlmU acetyltransferase: The reaction was performed with GlcN-1-P, AcCoA and different concentration of GlmU at 37oC for different time. The curve of velocity versus concentration of GlmU enzyme and the curve of concentration of product versus time were plotted. The results showed that the concentration of GlmU was 0.88μg/ml and the incubation time was 5 minutes within the initial velocity.GlmU uridyltransferase: The reaction was performed with GlcNAc-1-P, UTP, pyrophosphatase and different concentration of GlmU at 37oC for different time. The curve of velocity versus concentration of GlmU enzyme and the curve of concentration of product versus time were plotted. The results showed that the concentration of GlmU was 1.33μg/ml and the incubation time was 5 minutes within the initial velocity.(2) The optimal reaction conditions of GlmU were determined.The reaction was performed at different temperature and pH in the initial velocity. For aceytltransferase the optimal temperature of GlmU is 30oC and the optimal pH is 8.0, especially the presence of Mg2+ has no effect on the activity of acetyltranferase. For uridyltransferase the optimal temperature of GlmU is 42oC and the optimal pH is still 8.0, but Mg2+ is required for uridyltransferase activity and the optimal concentration is 20 mM.(3) The kinetic parameters of GlmU were determined.By using the above assays in the initial velocity and optimal condition one substrate was at a saturated state and the concentration of the other one was changed. Km and Vmax of GlmU were calculated by the double- reciprocal plot method. The Km and Vmax for AcCoA is 0.224±0.07 mM and 0.119±0.038 mMmin-1, respectively, for GlcN-1-P is 0.061±0.005 mM and 0.081±0.003 mMmin-1, respectively. The Km and Vmax for UTP is 0.024±0.0015 mM and 0.006±0.0001 mMmin-1, respectively, for GlcNAc-1- P is 0.044±0.005 mM and 0.0054±0.0002 mMmin-1.Conclusions:In this study, we overexpressed soluble M. tuberculosis GlmU in E.coli BL21(DE3) and purified GlmU protein for the further kinetic study. We developed accurate and reliable enzyme assays for studying bifunctional GlmU as well as for high-throughout screening inhibitors of GlmU. We studied the kinetics of GlmU and determined the optimal reaction condi- tions, Km and Vmax.