Identification of amino acids involved in catalytic process of M. tuberculosis GlmU acetyltransferase.
M. tuberculosis GlmU is a bifunctional enzyme with acetyltransferase activity in C-terminus and uridyltransferase activity in N-terminus, and it is involved in the biosynthesis of glycosyl donor UDP-N-acetylglucosamine (UDP-GlcNAc). The crystal structure of M. tuberculosis GlmU clearly determines the active site and catalytic mechanism of GlmU uridyltransferase domain but not succeed in GlmU acetyltransferase domain. Sequence comparison analysis revealed highly conserved amino acid residues in the C-terminus between M. tuberculosis GlmU and GlmU enzymes from other bacteria. To find the essential amino acids related to M. tuberculosis GlmU acetyltransferase activity, we substituted 10 conserved amino acids in the acetyltransferase domain of M. tuberculosis GlmU by site-directed mutagenesis. All the mutant GlmU proteins were largely expressed in soluble and purified by affinity chromatography. Enzyme assays showed that K362A, H374A, Y398A and W460A mutants abolished more than 90% activity of M. tuberculosis GlmU acetyltransferase and totally lost the affinity with two substrates, suggesting the potential substrate-binding functions. However, K403A, S416A, N456A and E458A mutants exhibited decreased GlmU acetyltransferase activity and lower kinetic parameters, probably responsible for substrate releasing by conformation shifting.
Department of Biochemistry and Molecular Biology, Dalian Medical Universtiy, Dalian, China.
Yan
Y
Zhou
Wendan
W
Yu
Qi
Q
Zheng
Yi
Y
Xin
Yufang
Y
Ma
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000096:biosynthesis
D000596:Amino Acids; D001426:Bacterial Proteins; C532100:GlmU protein, Mycobacterium tuberculosis; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014536:Uridine Diphosphate N-Acetylgalactosamine