Sevcík Jozef, Hostinová Eva, Solovicová Adriana, Gasperík Juraj, Dauter Zbigniew, Wilson Keith S
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia.
FEBS J. 2006 May;273(10):2161-71. doi: 10.1111/j.1742-4658.2006.05230.x.
Most glucoamylases (alpha-1,4-D-glucan glucohydrolase, EC 3.2.1.3) have structures consisting of both a catalytic and a starch binding domain. The structure of a glucoamylase from Saccharomycopsis fibuligera HUT 7212 (Glu), determined a few years ago, consists of a single catalytic domain. The structure of this enzyme with the resolution extended to 1.1 A and that of the enzyme-acarbose complex at 1.6 A resolution are presented here. The structure at atomic resolution, besides its high accuracy, shows clearly the influence of cryo-cooling, which is manifested in shrinkage of the molecule and lowering the volume of the unit cell. In the structure of the complex, two acarbose molecules are bound, one at the active site and the second at a site remote from the active site, curved around Tyr464 which resembles the inhibitor molecule in the 'sugar tongs' surface binding site in the structure of barley alpha-amylase isozyme 1 complexed with a thiomalto-oligosaccharide. Based on the close similarity in sequence of glucoamylase Glu, which does not degrade raw starch, to that of glucoamylase (Glm) from S. fibuligera IFO 0111, a raw starch-degrading enzyme, it is reasonable to expect the presence of the remote starch binding site at structurally equivalent positions in both enzymes. We propose the role of this site is to fix the enzyme onto the surface of a starch granule while the active site degrades the polysaccharide. This hypothesis is verified here by the preparation of mutants of glucoamylases Glu and Glm.
大多数葡糖淀粉酶（α-1,4-D-葡聚糖葡糖水解酶，EC 3.2.1.3）的结构都由一个催化结构域和一个淀粉结合结构域组成。几年前测定的来自扣囊复膜孢酵母HUT 7212（Glu）的葡糖淀粉酶结构由单一催化结构域构成。本文展示了该酶分辨率扩展至1.1 Å时的结构以及该酶与阿卡波糖复合物分辨率为1.6 Å时的结构。原子分辨率的结构除了具有高精度外，还清晰显示了低温冷却的影响，这表现为分子收缩和晶胞体积减小。在复合物结构中，结合了两个阿卡波糖分子，一个在活性位点，另一个在远离活性位点的位置，围绕着Tyr464弯曲，这类似于与硫代麦芽寡糖复合的大麦α-淀粉酶同工酶1结构中“糖钳”表面结合位点的抑制剂分子。基于不降解生淀粉的葡糖淀粉酶Glu与来自扣囊复膜孢酵母IFO 0111的生淀粉降解酶葡糖淀粉酶（Glm）在序列上的高度相似性，可以合理预期在这两种酶的结构等效位置存在远程淀粉结合位点。我们提出该位点的作用是在活性位点降解多糖时将酶固定在淀粉颗粒表面。本文通过制备葡糖淀粉酶Glu和Glm的突变体验证了这一假设。
