The kinetic characterization of Escherichia coli MurG using synthetic substrate analogues

Bacterial resistance to existing antibiotics poses a serious threat to human health. Because the peptidoglycan layer surrounding bacterial cells is essential for survival, the enzymes involved in peptidoglycan biosynthesis are attractive targets for the design of new antibiotics. Unfortunately, many of these enzymes are difficult to study because substrates to monitor enzymatic activity are either not available or not soluble under suitable assay conditions. These problems can be solved by utilizing synthetic alternative substrates. We recently reported the synthesis of a soluble substrate analogue for MurG, the enzyme that forms the beta-(1,4)-N-acetylglucosaminyl-N-acetylmuramyl pentapeptide subunit of peptidoglycan. Using this substrate analogue, we have been able to develop a direct assay to monitor the activity of the enzyme. We-now report the purification of Escherichia coli MurG and information on its kinetic properties and substrate requirements in the absence of membranes. This work lays the foundation for detailed mechanistic and structural investigations of this essential bacterial enzyme.