Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria

Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl tra nsferase (MurA) from many bacterial sources were analyzed. It was shown tha t whereas gram-negative bacteria have only one murA gene, gram-positive bac teria have two distinct genes encoding these enzymes which have possibly ar isen from gene duplication. The two murA genes of the gram-positive organis m Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organis m was viable despite losing one of its murA genes. However, when attempts w ere made to construct a double-deletion strain, no mutants were obtained. T his indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the o rganism. The two genes were further cloned and overexpressed, and the enzym es they encode were purified. Both enzymes catalyzed the transfer of enolpy ruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases, The cata lytic parameters of the two enzymes were similar, and they were both inhibi ted by the antibiotic fosfomycin.