Characterization and mutational analysis of two UDP-galactose 4-epimerases in Streptococcus pneumoniae TIGR4

Abstract

Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to cope with diseases caused by different serotypes of Streptococcus pneumoniae. UDP-galactose 4-epimerase (GalE) is an essential enzyme involved in polysaccharide synthesis. It is an important virulence factor in many bacterial pathogens. In this study, we found that two genes (galE_sp1 and galE_sp2) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalE_Sp1 and Gal_ESp2 were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalE^Sp2 was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetylgalactosamine (UDP-GalNAc). Interestingly, GalE_Sp2 had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalE_Sp1. The biochemical properties of GalE_Sp2 were studied. GalE_Sp2 was stable over a wide range of temperatures, between 30 and 70°C, at pH 8.0. The K86G substitution caused GalE_Sp2 to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalE_Sp2 resulted in only decreased activity toward UDP-GlcNAc and UDP-GalNAc. These results indicate that the Lys86 residue plays a critical role in the activity and substrate specificity of GalE_Sp2.