Characterization and Mutational Analysis of Two UDP-Galactose 4-Epimerases in Streptococcus pneumoniae TIGR4
Characterization and Mutational Analysis of Two UDP-Galactose 4-Epimerases in Streptococcus pneumoniae TIGR4
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DOI：
10.1134/s0006297918010054
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发表时间：
2018-01-01
影响因子：
2.8
通讯作者：
Chen, M.
中科院分区：
文献类型：
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作者：
Chen, L. L.;Han, D. L.;Chen, M.
目前肺炎球菌感染的临床治疗有许多局限性，并面临许多挑战。必须探索新的荚膜多糖结构以科普由不同血清型肺炎链球菌引起的疾病。UDP-半乳糖4-差向异构酶（GalE）是参与多糖合成的必需酶。它是许多细菌病原体的重要毒力因子。在本研究中，我们发现两个基因（galE（sp1）和galE（sp2））负责致病性S. pneumoniae TIGR4. GalE（Sp1）和GalE（Sp2）均显示催化UDP-葡萄糖（UDP-Glc）/UDP-半乳糖（UDP-Gal）的差向异构化，但仅GalESp 2显示催化UDP-N-乙酰葡糖胺（UDP-GlcNAc）/UDP-N-乙酰半乳糖胺（UDP-GalNAc）的差向异构化。有趣的是，GalE（Sp2）对UDP-Glc/UDP-Gal的差向异构酶活性比GalE（Sp1）高3倍。对GalE（Sp2）的生化特性进行了研究。GalE（Sp2）在30至70 ° C的宽温度范围内，在pH 8.0下是稳定的。K86 G取代导致GalE（Sp2）失去其对UDP-Glc和UDP-Gal的差向异构酶活性;然而，GalE（Sp2）中的取代C300 Y仅导致对UDP-GlcNAc和UDP-GalNAc的活性降低。这些结果表明Lys 86残基在GalE（Sp2）的活性和底物特异性中起关键作用。
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 GalE(Sp2) were shown to catalyze the epimerization of UDP-glucose (UDP-Glc)/UDP-galactose (UDP-Gal), but only GalESp2 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 degrees 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).