UDP-
N-acetyl-D-glucosamine (
UDP-GlcNAc) is an essential precursor of
peptidoglycan and the
rhamnose-GlcNAc linker region of mycobacterial cell wall. In Mycobacterium tuberculosis H37Rv genome, Rv1018c shows strong homology to the GlmU
protein involved in the formation of
UDP-GlcNAc from other bacteria. GlmU is a bifunctional
enzyme that catalyzes two sequential steps in
UDP-GlcNAc biosynthesis. Glucosamine-1-phosphate acetyl
transferase catalyzes the formation of
N-acetylglucosamine-1-phosphate, and
N-acetylglucosamine-1-phosphate uridylyltransferase catalyzes the formation of
UDP-GlcNAc. Since inhibition of
peptidoglycan synthesis often results in cell lysis, M.
tuberculosis GlmU is a potential anti-
tuberculosis (TB)
drug target. In this study we cloned M.
tuberculosis Rv1018c (glmU gene) and expressed soluble GlmU
protein in E. coli BL21(DE3). Enzymatic assays showed that M.
tuberculosis GlmU
protein exhibits both
glucosamine-1-phosphate acetyltransferase and
N-acetylglucosamine-1-phosphate uridylyltransferase activities. We also investigated the effect on Mycobacterium smegmatis when the activity of GlmU is fully removed or reduced via a genetic approach. The results showed that activity of GlmU is required for growth of M. smegmatis as the bacteria did not grow in the absence of active GlmU
enzyme. As the amount of functional GlmU
enzyme was gradually reduced in a temperature shift experiment, the M. smegmatis cells became non-viable and their morphology changed from a normal rod shape to stubby-rounded morphology and in some cases they lysed. Finally a microtiter plate based assay for GlmU activity with an OD340 read out was developed. These studies therefore support the further development of M.
tuberculosis GlmU
enzyme as a target for new
anti-tuberculosis drugs.