CHARACTERIZATION AND MUTATIONAL ANALYSIS OF TWO UDP-GALACTOSE 4-EPIMERASES IN <i>Streptococcuspneumoniae </i>TIGR4
Current clinical treatments for pneumococcal infections have many limitations and are faced with many challenges. New capsular polysaccharide structures must be explored to help prevent 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<sub>sp1 </sub> and galE<sub>sp2</sub>) are responsible for galactose metabolism in pathogenic S. pneumoniae TIGR4. Both GalE8<sub>p1</sub> and GalE<sub>8p2 </sub>were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalE<sub>8p2</sub> was shown to catalyze the epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc)/UDP-N-acetyl-galactosamine (UDP-GalNAc). Interestingly, GalE<sub>8p2</sub> had 3-fold higher epimerase activity toward UDP-Glc/UDP-Gal than GalE<sub>8p1</sub>. The biochemical properties of GalE<sub>8p2</sub> were studied. GalE<sub>8p2</sub> was stable over a wide range of temperatures, between 30 and 70 °C, at pH 8.0. The K86G substitution caused GalE<sub>8p2</sub> to lose its epimerase activity toward UDP-Glc and UDP-Gal; however, substitution C300Y in GalE<sub>8p2</sub> 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<sub>8p2</sub>.