Kinetic properties of Mycobacterium tuberculosis bifunctional GlmU |
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Authors: | Yan Zhou Yi Xin Shanshan Sha Yufang Ma |
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Affiliation: | (1) Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 W. Lushun South Road, Dalian, 116044, China;(2) Department of Biotechnology, Dalian Medical University, Dalian, 116044, China;(3) Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian, 116044, China; |
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Abstract: | The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors. |
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