Kinetic properties of Mycobacterium tuberculosis bifunctional GlmU
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ORIGINAL PAPER
Kinetic properties of Mycobacterium tuberculosis bifunctional GlmU Yan Zhou • Yi Xin • Shanshan Sha Yufang Ma
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Received: 18 May 2010 / Revised: 26 February 2011 / Accepted: 3 May 2011 / Published online: 19 May 2011 Ó Springer-Verlag 2011
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-1phosphate (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
Communicated by Jean-Luc Pernodet. Y. Zhou S. Sha Y. Ma (&) Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 W. Lushun South Road, Dalian 116044, China e-mail: [email protected] Y. Xin Department of Biotechnology, Dalian Medical University, Dalian 116044, China Y. Xin Y. Ma Liaoning Provincial Core Lab of Glycobiology and Glycoengineering, Dalian 116044, China
activities and the kinetic properties will facilitate highthroughput screening of GlmU inhibitors. Keywords M. tuberculosis GlmU Glucosamine-1-phosphate acetyltransferase N-acetyl-glucosamine-1-phosphate uridyltransferase Kinetics of GlmU
Introduction More attention is diverted to tuberculosis once again due to its threat from the yearly rising morbidity and mortality following the prevalence of human immunodeficiency virus (HIV) (Suchindran et al. 2009) or combining with the emergence of multi-drug resistant tuberculosis (MDR-TB) or extensively drug resistant tuberculosis (XDR-TB) (Migliori et al. 2008). In this case, the first line antibiotics gradually loss their leading role in treating TB infections. Exploring new targets and developing specific drugs will be urgently needed nowadays against the pervasiveness of tuberculosis. The integrity of cell wall structure closely correlates to the survival of Mycobacterium tuberculosis. Three kinds of macromolecules compose the mycobacterial cell wall core including mycolic acids, arabinogalactan (AG), and peptidoglycan (PG) (Brennan and Crick 2007). A disaccharide linker, D-N-GlcNAc-L-rhamnose (Brennan and Nikaido 1995; Brennan 2003), covalently attaches AG to PG maintaining the integrity of cell wall core significantly. Thus, UDP-N-acetylglucosamine (
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