Liu Feng, Tanner Martin E
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
J Biol Chem. 2006 Jul 28;281(30):20902-20909. doi: 10.1074/jbc.M602972200. Epub 2006 May 25.
The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon alpha-keto acid, pseudaminic acid. The presence of this posttranslational modification is absolutely required for assembly of functional flagella. Since motility is required for colonization, pseudaminic acid biosynthesis represents a virulence factor in these bacteria. Pseudaminic acid is generated from UDP-N-acetylglucosamine in five biosynthetic steps. The final step has been shown to involve the condensation of 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-Altdi-NAc) with phosphoenolpyruvate as catalyzed by the enzyme pseudaminic acid synthase, NeuB3. The 6-deoxy-AltdiNAc used in this process is generated from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This manuscript describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be k(cat) = 27 s(-1) and K(m) = 174 microm. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C-1 and to utilize a C-O bond cleavage mechanism during catalysis. These results, coupled with homology comparisons, suggest that the closest ancestors to the hydrolase are members of the metal-independent GT-B family of glycosyltransferases that include the enzyme MurG.
空肠弯曲菌和幽门螺杆菌等致病细菌中的鞭毛蛋白被九碳α-酮酸假氨基糖大量糖基化。这种翻译后修饰的存在是功能性鞭毛组装所绝对必需的。由于定殖需要运动性，假氨基糖生物合成是这些细菌中的一种毒力因子。假氨基糖由UDP-N-乙酰葡糖胺经五个生物合成步骤生成。已表明最后一步涉及由假氨基糖合酶NeuB3催化的2,4-二乙酰氨基-2,4,6-三脱氧-L-阿卓糖（6-脱氧-Altdi-NAc）与磷酸烯醇丙酮酸的缩合反应。此过程中使用的6-脱氧-AltdiNAc由其核苷酸连接形式UDP-6-脱氧-AltdiNAc通过一种水解酶的作用生成，该水解酶切割糖苷键并释放UDP。本文描述了一种UDP-6-脱氧-AltdiNAc水解酶，即来自空肠弯曲菌的PseG（Cj1312）的首次表征。该酶的活性与二价金属离子的存在无关，催化常数的值为k(cat)=27 s(-1)和K(m)=174 μM。该酶在C-1处对底物进行水解时立体化学发生完全反转，并在催化过程中利用C-O键断裂机制。这些结果与同源性比较相结合，表明该水解酶最接近的祖先属于不依赖金属的GT-B家族糖基转移酶，其中包括MurG酶。
