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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Binding of bivalent ions to actinomycete mannanase is accompanied by conformational change and is a key factor in its thermal stability
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Binding of bivalent ions to actinomycete mannanase is accompanied by conformational change and is a key factor in its thermal stability

机译：二价离子与放线菌甘露聚糖酶的结合伴随构象变化，是其热稳定性的关键因素

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摘要

The study aimed to define the key factors involved in the modulation of actinomycete mannanases. We focused on the roles of carbohydrate-binding modules (CBMs) and bivalent ions. To investigate the effects of these factors, two actinomycete mannanase genes were cloned from Streptomyces thermoluteus (StManII) and Streptomyces lividans (SlMan). CBMs fused to mannanase catalytic domains do not affect the thermal stability of the proteins. CBM2 of StManII increased the catalytic efficiency toward soluble-mannan and insoluble-mannan by 25%-36%, and CBM10 of SlMan increased the catalytic efficiency toward soluble-mannan by 40%-50%. Thermal stability of wild-type and mutant enzymes was enhanced by calcium and manganese. Thermal stability of SlMandC was also slightly enhanced by magnesium. These results indicated that bivalent ion-binding site responsible for thermal stability was in the catalytic domains. Thermal stability of mannanase differed in the kinds of bivalent ions. Isothermal titration calorimetry revealed that the catalytic domain of StManII bound bivalent ions with a Ka of 5.39 ?? 0.45 ?? 10 3-7.56 ?? 1.47 ?? 103 M- 1, and the catalytic domain of SlMan bound bivalent ions with a Ka of 1.06 ?? 0.34 ?? 103-3.86 ?? 0.94 ?? 103 M- 1. The stoichiometry of these bindings was consistent with one bivalent ion-binding site per molecule of enzyme. Circular dichroism spectrum revealed that the presence of bivalent ions induced changes in the secondary structures of the enzymes. The binding of certain bivalent ion responsible for thermal stability was accompanied by a different conformational change by each bivalent ion. Actinomycete mannanases belong to GHF5 which contained various hemicellulases; therefore, the information obtained from mannanases applies to the other enzymes. ? 2012 Elsevier B.V. All rights reserved.
机译：该研究旨在确定参与放线菌甘露聚糖酶调节的关键因素。我们专注于碳水化合物结合模块（CBM）和二价离子的作用。为了研究这些因素的影响，从嗜热链霉菌（StManII）和淡紫色链霉菌（SlMan）中克隆了两个放线菌甘露聚糖酶基因。与甘露聚糖酶催化结构域融合的CBM不会影响蛋白质的热稳定性。 StManII的CBM2将其对可溶性甘露聚糖和不溶性甘露聚糖的催化效率提高25％-36％，SlMan的CBM10将对可溶性甘露聚糖的催化效率提高40％-50％。钙和锰增强了野生型和突变型酶的热稳定性。镁也使SlMandC的热稳定性略有提高。这些结果表明负责热稳定性的二价离子结合位点在催化域中。甘露聚糖酶的热稳定性在二价离子的种类方面有所不同。等温滴定量热法显示，StManII的催化结构域结合二价离子，Ka为5.39 ??。 0.45 ?? 10 3-7.56 ?? 1.47 ?? 103 M-1和SlMan结合的二价离子的催化结构域，Ka为1.06 ??。 0.34 ?? 103-3.86 ?? 0.94 ?? 103 M-1。这些结合的化学计量与每个酶分子的一个二价离子结合位点一致。圆二色性光谱显示，二价离子的存在诱导了酶的二级结构的变化。负责热稳定性的某些二价离子的结合伴随有每个二价离子的不同构象变化。放线菌甘露聚糖酶属于GHF5，含有各种半纤维素酶。因此，从甘露聚糖酶获得的信息适用于其他酶。 ？ 2012 Elsevier B.V.保留所有权利。

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