A novel trehalase from Mycobacterium smegmatis -: purification, properties, requirements
A novel trehalase from Mycobacterium smegmatis -: purification, properties, requirements
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DOI：
10.1111/j.1742-4658.2007.05715.x
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发表时间：
2007-04-01
期刊：
影响因子：
5.4
通讯作者：
Elbein, Alan D.
中科院分区：
文献类型：
--
作者：
Carroll, J. David;Pastuszak, Irena;Elbein, Alan D.
海藻糖是葡萄糖(α，α-1，1-葡萄糖)的一种非还原二糖，对分枝杆菌的生长和生存是必不可少的。这些生物有三种不同的生物合成途径来产生海藻糖，而缺乏这三种途径的突变株需要在培养基中加入外源海藻糖才能生长。污垢分枝杆菌和结核分枝杆菌也有一种海藻糖酶，这种酶在控制细胞内海藻糖水平方面可能很重要。在这项研究中，我们报道了耻垢分枝杆菌海藻糖酶的纯化和性质，并与结核分枝杆菌的海藻糖酶进行了比较。虽然这两种酶的氨基酸序列有85%以上的同源性，并且都显示出绝对需要无机磷才能发挥活性，但耻垢分枝杆菌的酶也需要镁离子才能发挥活性，而结核分枝杆菌海藻糖酶不需要镁离子。在糖基水解酶中，对磷酸盐的需求是不寻常的，但我们没有发现任何证据表明反应中存在磷酸化裂解或任何磷酸化的中间产物。然而，由于无机磷酸盐似乎与海藻糖酶结合，并极大地提高了海藻糖酶的热稳定性，因此磷酸盐的功能可能涉及稳定蛋白质构象和/或启动蛋白质聚集。砷酸钠能在一定程度上替代磷酸钠的需要量，而无机焦磷酸盐和聚磷酸盐则有抑制作用。纯化的海藻糖酶在凝胶上显示单一的71 kDa条带，但活性酶在Sephracryl S-300柱的空隙中洗脱，表明其相对分子质量约为1500 kDa或由20个或更多亚基组成的多聚体。海藻糖酶对α，α-海藻糖高度专一，不能水解α，β-海藻糖或β，β-海藻糖，海藻糖二乙醇酸酯，或任何其他α-葡萄糖苷或β-葡萄糖苷。获得污垢分枝杆菌海藻糖酶阴性突变体的尝试没有成功，尽管删除其他海藻糖代谢酶已经产生了可行的突变体。这表明海藻糖酶是这些生物的一种必不可少的酶。该酶的最适pH为7.1，只要有无机磷和镁离子存在，该酶在各种缓冲液中都能发挥活性。在磷酸盐或砷盐存在下，海藻糖酶产生的唯一产物是葡萄糖。
Trehalose is a nonreducing disaccharide of glucose (alpha,alpha-1,1-glucosyl-glucose) that is essential for growth and survival of mycobacteria. These organisms have three different biosynthetic pathways to produce trehalose, and mutants devoid of all three pathways require exogenous trehalose in the medium in order to grow. Mycobacterium smegmatis and Mycobacterium tuberculosis also have a trehalase that may be important in controlling the levels of intracellular trehalose. In this study, we report on the purification and characterization of the trehalase from M. smegmatis, and its comparison to the trehalase from M. tuberculosis. Although these two enzymes have over 85% identity throughout their amino acid sequences, and both show an absolute requirement for inorganic phosphate for activity, the enzyme from M. smegmatis also requires Mg2+ for activity, whereas the M. tuberculosis trehalase does not require Mg2+. The requirement for phosphate is unusual among glycosyl hydrolases, but we could find no evidence for a phosphorolytic cleavage, or for any phosphorylated intermediates in the reaction. However, as inorganic phosphate appears to bind to, and also to greatly increase the heat stability of, the trehalase, the function of the phosphate may involve stabilizing the protein conformation and/or initiating protein aggregation. Sodium arsenate was able to substitute to some extent for the sodium phosphate requirement, whereas inorganic pyrophosphate and polyphosphates were inhibitory. The purified trehalase showed a single 71 kDa band on SDS gels, but active enzyme eluted in the void volume of a Sephracryl S-300 column, suggesting a molecular mass of about 1500 kDa or a multimer of 20 or more subunits. The trehalase is highly specific for alpha,alpha-trehalose and did not hydrolyze alpha,beta-trelalose or beta,beta-trehalose, trehalose dimycolate, or any other alpha-glucoside or beta-glucoside. Attempts to obtain a trehalase-negative mutant of M. smegmatis have been unsuccessful, although deletions of other trehalose metabolic enzymes have yielded viable mutants. This suggests that trehalase is an essential enzyme for these organisms. The enzyme has a pH optimum of 7.1, and is active in various buffers, as long as inorganic phosphate and Mg2+ are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.