UDP-N-acetylglucosamine enolpyruvyl transferase from Pseudomonas aeruginosa

• Published in: World journal of microbiology & biotechnology Vol. 26; no. 9; pp. 1623 - 1629
• Main Authors: Dube, Smita, Nanda, Kamna, Rani, Reema, Kaur, Namrata Jit, Nagpal, Jatin Kumar, Upadhyay, Dilip J, Cliffe, Ian A, Saini, Kulvinder Singh, Purnapatre, Kedar P
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01-09-2010; Springer Netherlands; Springer Nature B.V
• Subjects: Antibiotics; Applied Microbiology; Bacteria; Biochemistry; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Cloning; Drug resistance; E coli; Enolpyruvyl-UDP-N-acetylglucosamine; Environmental Engineering/Biotechnology; Enzymes; Escherichia coli; Fosfomycin; Genomics; High throughput screening; Laboratories; Life Sciences; Microbiology; MurA; Original Paper; Pathogens; Proteins; Pseudomonas aeruginosa; Reagents; Studies; UDP-N-acetylglucosamine; High throughput screening; UDP-N-acetylglucosamine; MurA; Enolpyruvyl-UDP-N-acetylglucosamine; Fosfomycin
• ISSN: 0959-3993; 1573-0972
• DOI: 10.1007/s11274-010-0338-2

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5-7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.