Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae

The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positiv e pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31 % identity with the MurB protein from E scherichia coli, and contains the catalytic residues, substrate-binding res idues and FAD-binding motif identified previously in the E. coli protein. T he gene was cloned into the pET28a + expression vector, and the 34.5 kDa pr otein that it encodes was overexpressed in E, coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insolubl e. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavop rotein, like its E. coli homologue, with a characteristic absorption at 463 nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine en olpyruvate with the concomitant oxidation of NADPH, and was characterized k inetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen pr ovides a good starting point for the discovery of antibacterial agents agai nst MurB.