Nowadays, two millions deaths each year caused by Mycobacterium tuberculosis has been considered to be a major public health threat. However, vaccine failed to provide protective immunity and any efforts to control tuberculosis were compromised as they evolved into stronger, more drugresistant forms. As we know, the cell wall of all Mycobacterium species is very waxy, hydrophobic, and thicker than other bacteria, the low permeability and resistance of cell wall substantially contributes to the defense of adverse factors. Thereby, the enzymes involved in the metabolic pathways of the cell wall are potential excellent targets for new anti-tuberculosis drugs. The mycobacterial cell wall consists of the mycolate and peptidoglycan layer held together by arabinogalactan layer. AG is attached to the muramic acid residue of the PG through a disaccharide linker, and the glycan of PG is a disaccharide repeat unit. UDP-N-acetylglucosamine is an important precursor for the synthesis of PG layer, and also a direct glycosyl donor for disaccharide linker, therefore, it perhaps plays a vital role in mycobacterial growth. In mycobacteria, UDP-GlcNAc is an important sugar donor for both formation of dissarchride linker and biosynthesis of peptidoglycan in mycobacterial cell wall, therefore, lacking UDP-GlcNAc could have effect on the structural integration of mycobacterial cell wall and further on their cell morphology. Ec GlmM has been well characterized as phosphoglucosamine mutase to catalyze the second step in the synthesis of E. coli UDP-GlcNAc, and Msm MSMEG_1556 protein and Mtb Rv3441c protein have significant homology to Ec GlmM. To detect the phosphoglucosamine mutase activity of Msm MSMEG_1556 protein and Mtb Rv3441c protein, it is required to acquire soluble protein. Mtb Rv3441c protein was produced by using pET16b vector, unfortunately, Mtb Rv3441c protein was insoluble. To avoid the formation of inclusion bodies, a cold-shock expression vector pCold II with the Rv3441c gene was constructed. This vector was designed to AZD6244 perform efficient protein expression utilizing promoter derived from cspA gene, which was one of the cold-shock gene. When the incubation temperature of E. coli host cells was reduced sufficiently, the growth is temporarily halted and almost of protein expression decrease, while expression of a group of proteins called “coldshock proteins” was specifically induced. A significant overproduction of soluble Mtb Rv3441c protein in E. coli BL21 was observed. The Msm MSMEG_1556 protein was also produced by using the same protocol. In our study, we also set up an enzyme assay for detection of phosphoglucosamine mutase activity. Compared with previous methods by autoradiography and HPLC. The activity of both Msm MSMEG_1556 protein and Mtb Rv3441c protein was dependent on the presence of Mg2+. Glc-1,6- diP was also required for activity. The purified proteins exhibited little activity without Glc-1,6-diP, however, phosphoglucosamine mutase activity was remarkably enhanced in the presence of this compound. The phosphorylated phosphoglucosamine mutase was assumed to be active. It is unclear that Glc-1,6-diP is a phosphorylating agent or activator. We attempt to co-crystallize Glc-1,6-diP and Mtb Rv3441c protein so as to reveal this mechanism in follow-up research. Mtb Rv3441c gene has been proved to be essential for the growth of cells by using Himar1-based transposon site hybridization methodology. To assess the effect of mutated glmM gene on cell growth, morphology, cell wall structure, etc., we used a model mycobacterial strain, M. smegmatis, to construct conditional MSMEG_1556 gene knockout strain LS2 by inserting kanR cassette.