Characterization of mycobacterial UDP-N-acetylglucosamine enolpyruvyle transferase (MurA).
The mycobacterial peptidoglycan has structure and biosynthetic pathways to similar those of other bacteria. UDP-N-acetylglucosamine enolpyruvyle transferase (MurA) catalyzes the first reaction in the biosynthesis of peptidoglycan. The MurA enzyme has been identified from various bacterial species, but the in-depth biochemical properties of mycobacterial MurA have not been characterized. In this study, both Mycobacterium tuberculosis MurA protein and Mycobacterium smegmatis MurA protein were overexpressed in Escherichia coli and purified by affinity chromatography. MurA activity was detected by HPLC. A colorimetric assay of MurA activity was also developed and the kinetic properties of Mtb MurA and Msm MurA were determined using this colorimetric assay. A conditional murA gene knockout strain was constructed by DNA homologous recombination. The disruption of murA in the genome of M. smegmatis led to loss of viability at a non-permissive temperature. Drastic morphological and structural alterations in the M. smegmatis murA knockout strain were observed by scanning electron microscopy and transmission electron microscopy. These results demonstrated that murA was an essential gene for growth of M. smegmatis. Therefore, MurA is a potential target for developing new anti-tuberculosis drugs.
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: xuliming_bio@126.com.
Liming
L
Xu
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: wdt1044@sina.com.
Dongting
D
Wu
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: liangliu2000@163.com.
Liang
L
Liu
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: jiaqi001.happy@163.com.
Qi
Q
Zheng
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: songyu84417@163.com.
Yu
Y
Song
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: yeli_2013yl@126.com.
Li
L
Ye
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: shanshan_s@126.com.
Shanshan
S
Sha
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China. Electronic address: kjwbl@163.com.
Jian
J
Kang
Department of Biotechnology, Dalian Medical University, Dalian, China; Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian, China. Electronic address: jimxin@hotmail.com.
Yi
Y
Xin
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, China; Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian, China. Electronic address: yufang_ma@hotmail.com.
Yufang
Y
Ma
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003124:Colorimetry / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D055786:Gene Knockout Techniques; D020043:Genes, Essential; D007700:Kinetics; D050296:Microbial Viability / Q000528:radiation effects; D008855:Microscopy, Electron, Scanning; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase
Cell wall; MurA; Mycobacteria; Peptidoglycan; UDP-N-acetylglucosamine enolpyruvyle transferase