Laboratory of Biochemistry, Bioengineering Department, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.
Proteins. 2011 Feb;79(2):365-75. doi: 10.1002/prot.22884.
Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.
家族 16 碳水化合物活性酶成员地衣芽孢杆菌 1,3-1,4-β-葡聚糖酶和杂种云杉木葡聚糖内转糖基酶（XET16-34）在结构上高度相关，但显示出不同的底物特异性。虽然第一种酶结合线性葡寡糖，但第二种酶结合支链木葡寡糖。这两种酶的先前工程化亲核突变体都是糖基合成酶，可催化糖基氟化物供体与糖苷受体之间的缩合。为了将糖基合成酶技术扩展到生产由支链木葡糖和线性葡聚糖的杂交体组成的设计寡糖，对 1,3-1,4-β-葡聚糖酶的负亚基进行了酶工程改造，以接受支链底物。去除 1,3-1,4-β-葡聚糖酶的主要环并替换为 XET16-34 以打开结合裂隙导致折叠蛋白，该蛋白仍保持一些β-葡聚糖水解酶活性，但相应的亲核突变体与线性或支链糖基供体均不显示糖基合成酶活性。接下来，突变 1,3-1,4-β-葡聚糖酶β-片层形成结合位点裂隙的点突变以类似于 XET16-34 残基。最终的嵌合蛋白获得了与木葡聚糖的结合亲和力，并且不与β-葡聚糖结合。因此，结合特异性已被重新设计，但亲和力低，并且嵌合酶的亲核突变体没有显示出糖基合成酶活性来产生目标杂合寡糖。X 射线晶体学的结构分析根据蛋白质结构的变化解释了这些结果，并强调了进一步引入所需活性的工程方法。
