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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• 作者：Yuya Kumagai ; Kayoko Kawakami ; Misugi Uraji ; Tadashi Hatanaka ; ^{hatanaka@bio-ribs.com}
• 关键词：ANS ; 1-anilinonaphthalene-8-sulfonic ; CBM ; carbohydrate-binding module ; CD ; circular dichroism ; DNS ; 3 ; 5-dinitrosalicylic acid ; GM ; konjac glucomannan ; ITC ; isothermal titration calorimetry ; IvMan ; ivory nut mannan ; LBG ; locust bean gum ; PIPES ; piperazine
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2013
• 出版时间：January, 2013
• 年：2013
• 卷：1834
• 期：1
• 页码：301-307
• 全文大小：780 K

文摘

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 ¡À 0.45 ¡Á 10³-7.56 ¡À 1.47 ¡Á 10³ M^? 1, and the catalytic domain of SlMan bound bivalent ions with a K_a of 1.06 ¡À 0.34 ¡Á 10³-3.86 ¡À 0.94 ¡Á 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.