Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College St, Toronto, ON M5S 3E5, Canada.
Biochem J. 2018 Dec 19;475(24):3963-3978. doi: 10.1042/BCJ20180763.
produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module (GH74) revealed a highly specific, processive -xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform - the first in GH74 extending from subsites -4 to +6 - and unique mobile active-site loops. Site-directed mutagenesis revealed that glycine-476 was uniquely responsible for the promiscuous backbone-cleaving activity of GH74; replacement with tyrosine, which is conserved in many GH74 members, resulted in exclusive hydrolysis at unbranched glucose units. Likewise, systematic replacement of the hydrophobic platform residues constituting the positive subsites indicated their relative contributions to the processive mode of action. Specifically, W347 (+3 subsite) and W348 (+5 subsite) are essential for processivity, while W406 (+2 subsite) and Y372 (+6 subsite) are not strictly essential, but aid processivity.
产生一种含有糖苷水解酶（GH）家族 74 模块（AIQ73809）的单一多模块酶。GH74 模块（GH74）的重组生产和特性表明，它是一种具有高度特异性、连续性的木葡聚糖酶，能够在支化和非支化位置水解多糖主链。为游离酶和寡糖复合物获得的 X 射线晶体结构证明了一个广泛的疏水结合平台 - 第一个在 GH74 中从亚位点 -4 延伸到 +6 - ，并且具有独特的可移动活性位点环。定点突变揭示甘氨酸-476 是 GH74 中混杂的骨架切割活性的独特原因；用酪氨酸替代，酪氨酸在许多 GH74 成员中保守，导致仅在非支化葡萄糖单元处水解。同样，构成正亚基的疏水平台残基的系统替换表明了它们对连续性作用模式的相对贡献。具体而言，W347（+3 亚基）和 W348（+5 亚基）对连续性是必需的，而 W406（+2 亚基）和 Y372（+6 亚基）不是必需的，但有助于连续性。
