Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden.
J Mol Biol. 2010 Apr 2;397(3):724-39. doi: 10.1016/j.jmb.2010.01.072. Epub 2010 Feb 6.
Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized-that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 A resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (alpha/beta)(8) domain similar to a triose phosphate isomerase barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second beta-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the -1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the -1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.
基于序列和系统发育分析，糖苷水解酶（GH）家族 3 可分为几个不同的簇，这些簇在其一级序列的长度上有所不同。然而，关于 GH3 代表的结构数据仍然很少，因为只有三种结构已知，并且只有一种结构得到了彻底的表征——来自大麦的外切水解酶。为了更深入地了解 GH3 家族的结构，我们测定了 Thermotoga neapolitana 耐热β-葡萄糖苷酶的晶体结构，该酶在环保型工业生物合成方面具有重要的潜在应用价值，分辨率为 2.05Å。对选定的活性位点突变体进行了动力学表征，并呈现了突变体 D242A 的 2.1Å分辨率结构。来自 Th.neapolitana 的 Bgl3B 是 GH3 葡萄糖苷酶的第一个具有三结构域结构的例子。它由一个类似于三磷酸甘油醛异构酶桶的（α/β）（8）结构域、一个五链α/β三明治结构域（两者对于活性位点的组织都很重要）和一个未知功能的 C 末端纤维连接蛋白 III 结构域组成。值得注意的是，三磷酸甘油醛异构酶桶结构域的第二个β-链的方向相反，这对活性位点的形状有影响。活性位点位于结构同源酶的 1 号和 2 号结构域的界面上，比结构同源酶的相应位点更向溶剂开放，只有-1 位点得到了很好的定义。结构与活性位点变体的动力学研究相结合，可确定必需的催化残基（亲核试剂 D242 和酸碱 E458），以及-1 亚位的其他残基，包括 D58 和 W243，通过突变，证明它们对底物容纳/相互作用很重要。纤维连接蛋白 III 结构域的位置排除了该结构域直接参与识别小分子底物的可能性，尽管它可能参与了酶在大聚合底物上的固定以及热稳定性。
