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首页> 外文期刊>The FEBS journal >A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements
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A novel trehalase from Mycobacterium smegmatis - purification, properties, requirements

机译：耻垢分枝杆菌的新型海藻糖酶-纯化，性质，要求

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摘要

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 Mg(2+) for activity, whereas the M. tuberculosis trehalase does not require Mg(2+). 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 Mg(2+) are present. Glucose was the only product produced by the trehalase in the presence of either phosphate or arsenate.
机译：海藻糖是葡萄糖的一种非还原性二糖（α，α-1，1-葡萄糖基葡萄糖），对分枝杆菌的生长和存活至关重要。这些生物具有三种不同的生物合成途径来产生海藻糖，而缺乏所有这三种途径的突变体则需要培养基中的外源海藻糖才能生长。耻垢分枝杆菌和结核分枝杆菌也具有海藻糖酶，在控制细胞内海藻糖水平中可能很重要。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表征，以及与结核分枝杆菌海藻糖酶的比较。尽管这两种酶在其整个氨基酸序列中具有超过85％的同一性，并且都显示出绝对需要无机磷酸盐才能发挥活性，但耻垢分枝杆菌的酶也需要Mg（2+）才能发挥活性，而结核分枝杆菌海藻糖酶确实需要Mg（2+）不需要Mg（2+）。在糖基水解酶中对磷酸盐的需求是不同寻常的，但是我们找不到任何证据表明存在磷解作用或反应中存在任何磷酸化的中间体。但是，由于无机磷酸盐似乎与海藻糖酶结合并极大地增加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/或引发蛋白质聚集。砷酸钠能够在一定程度上替代磷酸钠的需求，而无机焦磷酸盐和多磷酸盐具有抑制作用。纯化的海藻糖酶在SDS凝胶上显示单个71 kDa条带，但活性酶在Sephracryl S-300色谱柱的空隙体积中洗脱，表明分子量约为1500 kDa或20个或更多亚基的多聚体。海藻糖酶对α，α-海藻糖具有高度特异性，并且不水解α，β-海藻糖或β，β-海藻糖，海藻糖二甲酸酯或任何其他α-葡萄糖苷或β-葡萄糖苷。尽管获得了其他海藻糖代谢酶的缺失，但仍无法获得耻垢分枝杆菌海藻糖酶阴性突变体的尝试。这表明海藻糖酶是这些生物的必需酶。该酶的最适pH值为7.1，只要存在无机磷酸盐和Mg（2+），它在各种缓冲液中均具有活性。葡萄糖是海藻糖酶在磷酸盐或砷酸盐存在下产生的唯一产物。

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