Key Laboratory of Biotechnology of Antibiotics, Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tian Tan, Beijing, China.
J Appl Microbiol. 2010 May;108(5):1544-51. doi: 10.1111/j.1365-2672.2009.04550.x. Epub 2009 Sep 30.
Ste15 and ste22 present in the Ebosin biosynthesis gene cluster (ste) were previously shown to function in Ebosin biosynthesis and both of the protein products are predicted to be glycosyltransferases. In this study, their biochemical activities were confirmed.
ste15 and ste22 were cloned and expressed in Escherichia coli. With a continuous coupled spectrophotometric assay and using the purified proteins, we now demonstrated that the protein Ste15 has the ability of catalysing the transfer of glucose specifically from UDP-glucose to an Ebosin precursor that lacks glucose, the lipid carrier located in the cytoplasmic membrane of the gene ste15 disrupt mutant Streptomyces sp. 139 (ste15(-)). The protein Ste22 can catalyse the transfer of rhamnose specifically from TDP-rhamnose to an Ebosin precursor that lacks rhamnose, a lipophilic carrier in the cytoplasmic membrane of the gene ste22 disrupt mutant Streptomyces sp. 139 (ste22(-)).
The gene product of ste15 was identified to be a glucosyltransferase, and the protein encoded by ste22 was found to be a rhamnosyltransferase.
Both of two enzymes play essential roles in the formation of repeating units of sugars during Ebosin biosynthesis. These are the first glucosyltransferase and rhamnosyltransferase in the biosynthesis of a Streptomyces exopolysaccharide to be characterized.
先前研究表明，Ebosin 生物合成基因簇（ste）中的 Ste15 和 Ste22 参与 Ebosin 生物合成，这两种蛋白质产物都被预测为糖基转移酶。本研究旨在证实它们的生化活性。
克隆并在大肠杆菌中表达 ste15 和 ste22。通过连续偶联分光光度法，使用纯化的蛋白质，我们现在证明了 Ste15 蛋白具有催化特定从 UDP-葡萄糖向缺乏葡萄糖的 Ebosin 前体转移葡萄糖的能力，该前体位于基因 ste15 缺失突变株 Streptomyces sp. 139（ste15(-)）的细胞质膜中的脂质载体上。Ste22 蛋白可以催化特定从 TDP-鼠李糖向缺乏鼠李糖的 Ebosin 前体转移鼠李糖，该前体位于基因 ste22 缺失突变株 Streptomyces sp. 139（ste22(-)）的细胞质膜中的亲脂性载体上。
鉴定出 ste15 基因产物为葡萄糖基转移酶，而 ste22 编码的蛋白被发现为鼠李糖基转移酶。
这两种酶在 Ebosin 生物合成过程中糖重复单元形成中都起着至关重要的作用。这是首次对链霉菌胞外多糖生物合成中的葡萄糖基转移酶和鼠李糖基转移酶进行了表征。
