Kumagai Yuya, Kawakami Kayoko, Uraji Misugi, Hatanaka Tadashi
Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences (RIBS), Okayama, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
Biochim Biophys Acta. 2013 Jan;1834(1):301-7. doi: 10.1016/j.bbapap.2012.08.011. Epub 2012 Aug 17.
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 K(a) of 5.39±0.45×10(3)-7.56±1.47×10(3)M(-1), and the catalytic domain of SlMan bound bivalent ions with a K(a) of 1.06±0.34×10(3)-3.86±0.94×10(3)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.
该研究旨在确定参与调节放线菌甘露聚糖酶的关键因素。我们重点关注碳水化合物结合模块（CBMs）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）和变铅青链霉菌（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CBMs不会影响蛋白质的热稳定性。StManII的CBM2使对可溶性甘露聚糖和不溶性甘露聚糖的催化效率提高了25%-36%，SlMan的CBM10使对可溶性甘露聚糖的催化效率提高了40%-50%。钙和锰增强了野生型和突变型酶的热稳定性。镁也略微增强了SlMandC的热稳定性。这些结果表明，负责热稳定性的二价离子结合位点位于催化结构域中。甘露聚糖酶的热稳定性因二价离子种类而异。等温滴定量热法显示，StManII的催化结构域结合二价离子的解离常数K(a)为5.39±0.45×10(3)-7.56±1.47×10(3)M(-1)，SlMan的催化结构域结合二价离子的解离常数K(a)为1.06±0.34×10(3)-3.86±0.94×10(3)M(-1)。这些结合的化学计量与每个酶分子一个二价离子结合位点一致。圆二色光谱显示，二价离子的存在诱导了酶二级结构的变化。负责热稳定性的某些二价离子的结合伴随着每种二价离子不同的构象变化。放线菌甘露聚糖酶属于GHF5，其中包含各种半纤维素酶；因此，从甘露聚糖酶获得的信息适用于其他酶。
