Comparison of the D-glutamate-adding enzymes from selected gram-positive and gram-negative bacteria

The biochemical properties of the D-glutamate-adding enzymes (MurD) from Es cherichia coli, Haemophilus influenzae, Enterococcus faecalis, and Staphylo coccus aureus were investigated to detect any differences in the activity o f this enzyme between gram-positive and gram-negative bacteria. The genes ( murD) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein-MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolyt ic treatments of the fusion proteins with factor Xa regenerated the individ ual MurD proteins. It was found that these fusion proteins retain D-glutama te-adding activity and have K-m and V-max, values similar to those of the r egenerated MurDs, except for the H. influenzae enzyme. Substrate inhibition by UDP-N-acetylmuramyl-L-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 mu M for E. coli and H. influenzae Mu rD, respectively. Such substrate inhibition was not observed with the E. fa ecalis and S. aureus enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require m onocations such as NH4+ and/or K+, but not Na+, for optimal activity, while anions such as Cl- and SO42- had no effect on the enzyme activities. The a ctivities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg2+ for the ligase activity and exhibited optimal activities around pH 8. These di fferences observed between the gram-positive and gram-negative MurDs indica ted that the two gram-negative bacteria may apply a more stringent regulati on of cell wall biosynthesis at the early stage of peptidoglycan biosynthes is pathway than do the two gram-positive bacteria. Therefore, the MurD-cata lyzed reaction may constitute a fine-tuning step necessary for the gramnega tive bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.