Ning B, Elbein A D
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA.
Arch Biochem Biophys. 1999 Feb 15;362(2):339-45. doi: 10.1006/abbi.1998.1053.
The enzyme that catalyzes the formation of GDP-d-mannose from GTP and alpha-d-mannose-1-P was purified about 2300-fold to near homogeneity from the soluble fraction of Mycobacterium smegmatis. At the final stage of purification, a major protein band of 37 kDa was observed and this band was specifically labeled, and in a concentration-dependent manner, by the photoaffinity probe 8-N3-GDP[32P]-d-mannose. The purified enzyme was stable for several months when kept in the frozen state. The 37-kDa band was subjected to protein sequencing and one peptide sequence of 25 amino acids showed over 80% identity to GDP-mannose pyrophosphorylases of pig liver and Saccharomyces cerevesiae. In contrast to some other bacterial GDP-mannose pyrophosphorylases, the mycobacterial enzyme was not multifunctional and did not have phosphomannose isomerase or phosphoglucose isomerase activity. Also, in contrast to the pig liver enzyme which uses mannose-1-P or glucose-1-P plus GTP to synthesize either GDP-mannose or GDP-glucose, the mycobacterial enzyme was specific for mannose-1-P as the sugar phosphate substrate. The enzyme was also relatively specific for GTP as the nucleoside triphosphate substrate. ITP was about 18% as effective as GTP, but ATP, CTP, and UTP were inactive. The activity of the enzyme was inhibited by GDP-glucose and glucose-1-P, although neither was a substrate for this enzyme. The pH optimum for the enzyme was 8.0, and Mg2+ was the best cation with optimum activity at about 5 mM. This enzyme is important for producing the activated form of mannose for formation of cell wall lipoarabinomannan and various mannose-containing glycolipids and polysaccharides.
从耻垢分枝杆菌的可溶性组分中纯化出催化由GTP和α-D-甘露糖-1-磷酸形成GDP-D-甘露糖的酶，纯化倍数约为2300倍，达到近乎均一的程度。在纯化的最后阶段，观察到一条37 kDa的主要蛋白条带，该条带被光亲和探针8-N3-GDP[32P]-D-甘露糖特异性标记，且呈浓度依赖性。纯化后的酶在冷冻状态下可稳定保存数月。对这条37 kDa的条带进行蛋白质测序，得到一个25个氨基酸的肽段序列，与猪肝和酿酒酵母的GDP-甘露糖焦磷酸化酶有超过80%的同一性。与其他一些细菌的GDP-甘露糖焦磷酸化酶不同，分枝杆菌的这种酶不是多功能的，不具有磷酸甘露糖异构酶或磷酸葡萄糖异构酶活性。此外，与使用甘露糖-1-磷酸或葡萄糖-1-磷酸加GTP来合成GDP-甘露糖或GDP-葡萄糖的猪肝酶不同，分枝杆菌的这种酶对甘露糖-1-磷酸作为磷酸糖底物具有特异性。该酶对GTP作为核苷三磷酸底物也相对具有特异性。ITP的效力约为GTP的18%，但ATP、CTP和UTP无活性。尽管GDP-葡萄糖和葡萄糖-1-磷酸都不是该酶的底物，但它们会抑制该酶的活性。该酶的最适pH为8.0，Mg2+是最佳阳离子，在约5 mM时活性最佳。这种酶对于产生用于形成细胞壁脂阿拉伯甘露聚糖以及各种含甘露糖的糖脂和多糖的活化形式的甘露糖很重要。
