Functional Characterization of UDP-Glucose:Undecaprenyl-Phosphate Glucose-1-Phosphate Transferases of Escherichia coli and Caulobacter crescentus
Functional Characterization of UDP-Glucose:Undecaprenyl-Phosphate Glucose-1-Phosphate Transferases of Escherichia coli and Caulobacter crescentus
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DOI：
10.1128/jb.06052-11
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发表时间：
2012-05-01
影响因子：
3.2
通讯作者：
Valvano, Miguel A.
中科院分区：
文献类型：
--
作者：
Patel, Kinnari B.;Toh, Evelyn;Valvano, Miguel A.
预计大肠杆菌 K-12 WcaJ 和新月柄杆菌 HfsE、PssY 和 PssZ 酶分别启动可拉酸 (CA) 胶囊和固着多糖的合成。这些蛋白质属于原核膜酶家族，催化连接己糖-L-磷酸与十一异戊烯基磷酸酯 (Und-P) 的磷酸酐键的形成。在这项研究中，大肠杆菌 K-12 weal 突变体的体内互补测定表明，WcaJ 和 PssY 可以补充 CA 合成。此外，WcaJ 可以恢复 C. crescentus 的固着力生产。体外转移酶测定表明，WcaJ 和 PssY 均利用 UDP-葡萄糖，但不利用 UDP-半乳糖。然而，在缺乏 WbaP O 抗原启动半乳糖基转移酶的鼠伤寒沙门氏菌菌株中，与 WcaJ 或 PssY 的互补导致产生 O 抗原。脂多糖 (LPS) 的气相色谱-质谱 (GC-MS) 分析揭示了 CA 和 O 抗原分子与脂质 A 核心寡糖 (OS) 的附着。因此，虽然 UDP-葡萄糖是 WcaJ 和 PssY 的首选底物，但这些酶也可以利用 UDP-半乳糖。 WcaJ 和 PssY 的这种意想不到的特征可能有助于绘制负责这些或类似酶的核苷酸二磷酸特异性的特定残基。此外，沙门氏菌中 O 抗原合成的重建、大肠杆菌中 CA 胶囊的合成以及固着物合成提供了高灵敏度的生物测定，以检查异源蛋白质的糖 1-磷酸转移酶特异性。
Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-l-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 weal mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.