Identification and Characterization of a MurA, UDP-N-Acetylglucosamine Enolpyruvyl Transferase from Cariogenic Streptococcus Mutans

Abstract

Streptococcus mutans (S. mutans) is the primary etiological agent of human dental caries. A gene (SMU_1525) encoding MurA was identified in S. mutans UA159 (ATCC700610). The deduced amino-acid sequence has homology (51% identity) with E. coli MurA protein (AAC76221). In this study, we cloned S. mutans murA (SMU_1525) gene, expressed soluble recombinant MurA protein in E. coli BL21(DE3). The recombinant protein was purified by affinity chromatography. The molecular weight of expressed protein was about 45.6 kD. The activity of the protein was identified by high-pressure liquid chromatography (HPLC) and was shown to have UDP-N-acetylglucosamine enolpyruvyl transferase activity. Then one microtiter plate based colourimetric assay for S. mutans MurA activity was developed. The optimal temperature and pH of the purified enzyme were 37°C and 7.5, respectively. The K_m for UDP-GlcNAc was 0.120 mM and for PEP was 0.086 mM. The V_max for UDP-GlcNAc was 0.048 mM min^-1 mg^-1 and for PEP was 0.098 mM min^-1 mg^-1. The activity of S. mutans MurA was inhibited by the fosfomycin, known MurA inhibitor.