Isomaltooligosaccharide-binding structure of Paenibacillus sp. 598K cycloisomaltooligosaccharide glucanotransferase
Isomaltooligosaccharide-binding structure of Paenibacillus sp. 598K cycloisomaltooligosaccharide glucanotransferase
复制标题
类芽孢杆菌属的低聚异麦芽糖结合结构。
作者：
Z. Fujimoto;N. Kishine;N. Suzuki;R. Suzuki;Daiki Mizushima;M. Momma;K. Kimura;K. Funane
类芽孢杆菌属598 K环异麦芽糖寡糖葡聚糖转移酶（CITase）是糖苷水解酶家族66（GH 66）的成员，催化葡聚糖的分子内转葡糖基化以产生具有七个或更多个聚合度的CI。为了阐明酶的环化反应和产物特异性，我们测定了PsCITase的晶体结构。PsCITase的核心结构由四个结构域组成：一个催化（β/α）8-结构域和三个β-结构域。家族35碳水化合物结合模块（类芽孢杆菌属物种598 K柠檬酸酶的第一个CBM 35区域，（PsCBM 35 -1））插入催化结构域并从催化结构域突出。通过用环异麦芽七糖浸泡晶体制备的PsCITase的配体络合物结构在三个位点产生结合糖：在催化裂缝中，在PsCBM 35 -1结构域的接合处和在PsCBM 35 -1的环区域。在催化位点中，观察到浸泡的环异麦芽七糖为线性异麦芽七糖，推测其为环异麦芽七糖被酶水解的产物，并占据亚位点-7至-1。在亚位点-7之外，观察到另一种异麦芽低聚糖的三个葡萄糖部分，认为这些位置是远端亚位点-13至-11。第三个结合位点是PsCBM 35 -1环区的典型糖结合位点，其中结合了浸泡的环异麦芽七糖。结构分析表明，催化结构域与PsCBM 35 -1之间的凹面在环化反应中起着引导长链底物的作用。
Paenibacillus sp. 598K cycloisomaltooligosaccharide glucanotransferase (CITase), a member of glycoside hydrolase family 66 (GH66), catalyses the intramolecular transglucosylation of dextran to produce CIs with seven or more degrees of polymerization. To clarify the cyclization reaction and product specificity of the enzyme, we determined the crystal structure of PsCITase. The core structure of PsCITase consists of four structural domains: a catalytic (β/α)8-domain and three β-domains. A family 35 carbohydrate-binding module (first CBM35 region of Paenibacillus sp. 598K CITase, (PsCBM35-1)) is inserted into and protrudes from the catalytic domain. The ligand complex structure of PsCITase prepared by soaking the crystal with cycloisomaltoheptaose yielded bound sugars at three sites: in the catalytic cleft, at the joint of the PsCBM35-1 domain and at the loop region of PsCBM35-1. In the catalytic site, soaked cycloisomaltoheptaose was observed as a linear isomaltoheptaose, presumably a hydrolysed product from cycloisomaltoheptaose by the enzyme and occupied subsites –7 to –1. Beyond subsite –7, three glucose moieties of another isomaltooiligosaccharide were observed, and these positions are considered to be distal subsites –13 to –11. The third binding site is the canonical sugar-binding site at the loop region of PsCBM35-1, where the soaked cycloisomaltoheptaose is bound. The structure indicated that the concave surface between the catalytic domain and PsCBM35-1 plays a guiding route for the long-chained substrate at the cyclization reaction.