Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase
Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase
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分枝杆菌 GlmU、双功能葡萄糖胺-1-磷酸乙酰转移酶和 N-乙酰葡萄糖胺-1-磷酸尿苷基转移酶的表达、本质和微量滴定板测定
DOI：
10.1016/j.biocel.2008.05.003
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发表时间：
2008-01-01
影响因子：
4
通讯作者：
Ma, Yufang
中科院分区：
文献类型：
--
作者：
Zhang, Wenli;Jones, Victoria C.;Ma, Yufang
UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor of pepticloglycan 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 pepticloglycan 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. (C) 2008 Elsevier Ltd. All rights reserved.
