Kinetic properties of wild-type and C117D mutant UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Haemophilus influenzae

摘要

In this study, the kinetic properties of wild-type and C117D mutant H. influenzae MurA (Hi MurA), which catalyzes the first reaction in the biosynthetic pathway of the cell wall, were characterized. Purified recombinant Hi MurA was active at pH values ranging from pH 5.5 to pH 10, and its Km (UNAG), Km (PEP), and kcat values were measured to be 31 μM, 24 μM, and 210 min−1, respectively. Hi MurA activity was effectively inhibited by fosfomycin with an IC50 value of 60 μM. Hi MurA contains a cysteine residue (C117) at the loop region near the PEP binding, whereas MurA from fosfomycin resistant Mycobaterium tuberculosis or Chlamydia trachomatis contain an aspartate residue instead of the cysteine at the corresponding site. Aspartate substitution of Cys117 in Hi MurA shifted its optimum pH from 7.8 to 6.0. In addition, the Km values for UNAG and PEP were increased to 160 μM and 150 μM, respectively, and the kcat value was significantly reduced to 41 min−1. Furthermore, the C117D mutant form of Hi MurA was not inhibited by 1 mM fosfomycin. These results indicate that the Cys117 of Hi MurA is the binding site of fosfomycin and plays an important role in the fast turnover of the catalytic reaction.