Identification of a novel UDP- N -acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio fischeri that confers hig
- PDF / 373,234 Bytes
- 5 Pages / 595 x 791 pts Page_size
- 37 Downloads / 170 Views
ORIGINAL PAPER
IdentiWcation of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Vibrio Wscheri that confers high fosfomycin resistance in Escherichia coli Sanath Kumar · Ammini Parvathi · Ricardo L. Hernandez · Kathleen M. Cadle · Manuel F. Varela
Received: 27 November 2008 / Revised: 26 January 2009 / Accepted: 19 February 2009 / Published online: 11 March 2009 © Springer-Verlag 2009
Abstract MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identiWed, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio Wscheri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. Wscheri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. Wscheri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 g/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identiWed as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and puriWed by aYnity chromatography. MurA of V. Wscheri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.
Communicated by Jorge Membrillo-Hernández. S. Kumar · R. L. Hernandez · K. M. Cadle · M. F. Varela (&) Department of Biology, Eastern New Mexico University, Roosevelt Hall, Room 101, Station 33, Portales, NM 88130, USA e-mail: [email protected] A. Parvathi Department of Biological Oceanography, National Institute of Oceanography Regional Centre (CSIR), Kochi 682 018, India
Keywords MurA · Fosfomycin · Antibiotic resistance · Vibrio Wscheri · MIC
Introduction The cell wall of bacteria is composed of alternating units of N-acetylglucosamine and N-acetylmuramic acid interconnected by penta-peptide cross links. The peptidoglycan cell wall determines the cell shape and provides the essential rigidity to withstand the high internal osmotic pressure. The Wrst committed step in the synthesis of bacterial cell wall takes place in the cytoplasm, which involves the addition of enolpyruvate from phosphoenolpyruvate (PEP) to the 3⬘-hydroxyl group of UDP-N-acetylglucosamine (UDP-NAG) with the release of inorganic phosphate (Bugg and Walsh 1992). This reaction is catalyzed by the enzyme UDP-NAG enolpyruvyl transferase (MurA) (EC 2.5.1.7). MurA is an essential enzyme in Escherichia coli, as its inactivation is lethal to the organism due to the loss of cell integrity and susceptibility to osmotic lysis (Brown et al. 1995). This enzyme is a target for the antibiotic fosfomycin which inactivates the enzyme by irreversibly binding to the enzyme formi
Data Loading...
