Madhuprakash Jogi, Dalhus Bjørn, Rani T Swaroopa, Podile Appa Rao, Eijsink Vincent G H, Sørlie Morten
Department of Chemistry, Biotechnology and Food Science , Norwegian University of Life Sciences (NMBU) , P.O. Box 5003, 1432 Ås , Norway.
Department of Plant Sciences , School of Life Sciences, University of Hyderabad , Gachibowli, Hyderabad , India.
Biochemistry. 2018 Jul 24;57(29):4325-4337. doi: 10.1021/acs.biochem.8b00381. Epub 2018 Jul 11.
Understanding features that determine transglycosylation (TG) activity in glycoside hydrolases is important because it would allow the construction of enzymes that can catalyze controlled synthesis of oligosaccharides. To increase TG activity in two family 18 chitinases, chitinase D from Serratia proteamaculans ( SpChiD) and chitinase A from Serratia marcescens ( SmChiA), we have mutated residues important for stabilizing the reaction intermediate and substrate binding in both donor and acceptor sites. To help mutant design, the crystal structure of the inactive SpChiD-E153Q mutant in complex with chitobiose was determined. We identified three mutations with a beneficial effect on TG activity: Y28A (affecting the -1 subsite and the intermediate), Y222A (affecting the intermediate), and Y226W (affecting the +2 subsite). Furthermore, exchange of D151, the middle residue in the catalytically important DXDXE motif, to asparagine reduced hydrolytic activity ≤99% with a concomitant increase in apparent TG activity. The combination of mutations yielded even higher degrees of TG activity. Reactions with the best mutant, SpChiD-D151N/Y226W/Y222A, led to rapid accumulation of high levels of TG products that remained stable over time. Importantly, the introduction of analogous mutations at the same positions in SmChiA (Y163A equal to Y28A and Y390F similar to Y222A) had similar effects on TG efficiency. Thus, the combination of the decreasing hydrolytic power, subsite affinity, and stability of intermediate states provides a powerful, general strategy for creating hypertransglycosylating mutants of retaining glycoside hydrolases.
了解决定糖苷水解酶中转糖基化（TG）活性的特征非常重要，因为这将有助于构建能够催化寡糖可控合成的酶。为了提高两种18家族几丁质酶（粘质沙雷氏菌的几丁质酶D（SpChiD）和粘质沙雷氏菌的几丁质酶A（SmChiA））的TG活性，我们对供体和受体位点中稳定反应中间体和底物结合的重要残基进行了突变。为了辅助突变设计，我们测定了与壳二糖复合的无活性SpChiD-E153Q突变体的晶体结构。我们鉴定出三个对TG活性有有益影响的突变：Y28A（影响-1亚位点和中间体）、Y222A（影响中间体）和Y226W（影响+2亚位点）。此外，将催化重要的DXDXE基序中的中间残基D151替换为天冬酰胺，水解活性降低≤99%，同时表观TG活性增加。突变的组合产生了更高程度的TG活性。与最佳突变体SpChiD-D151N/Y226W/Y222A的反应导致高水平TG产物的快速积累，且这些产物随时间保持稳定。重要的是，在SmChiA的相同位置引入类似突变（Y163A等同于Y28A，Y390F类似于Y222A）对TG效率有类似影响。因此，降低水解能力、亚位点亲和力和中间态稳定性的组合为创建保留糖苷水解酶的超转糖基化突变体提供了一种强大的通用策略。
