Ortiz-Soto Maria Elena, Possiel Christian, Görl Julian, Vogel Andreas, Schmiedel Ramona, Seibel Jürgen
Institute of Organic Chemistry, University of Würzburg, Würzburg, Am Hubland 97074, Germany.
c-LEcta GmbH, Leipzig, Perlickstr. 5, 04103, Germany.
Glycobiology. 2017 Aug 1;27(8):755-765. doi: 10.1093/glycob/cwx050.
Bacterial levansucrases produce β(2,6)-linked levan-type polysaccharides using sucrose or sucrose analogs as donor/acceptor substrates. However, the dominant reaction of Bacillus megaterium levansucrase (Bm-LS) is hydrolysis. Single domain levansucrases from Gram-positive bacteria display a wide substrate-binding pocket with open access to water, challenging engineering for transfructosylation-efficient enzymes. We pursued a shift in reaction specificity by either modifying the water distribution in the active site or the coordination of the catalytic acid/base (E352) and the nucleophile (D95), thus affecting the fructosyl-transfer rate and allowing acceptors other than water to occupy the active site. Two serine (173/422) and two water-binding tyrosine (421/439) residues located in the first shell of the catalytic pocket were modified. Library variants of S173, Y421 and S422, which coordinate the position of D95 and E352, show increased transfructosylation (30-200%) and modified product spectra. Substitutions at position 422 have a higher impact on sucrose affinity, while changes at position 173 and 421 have a strong effect on the overall catalytic rate. As most retaining glycoside hydrolases (GHs) Bm-LS catalyzes hydrolysis and transglycosylation via a double displacement reaction involving two-transition states (TS1 and TS2). Hydrogen bonds of D95 with the side chains of S173 and S422 contribute a total of 2.4 kcal mol-1 to TS1 stabilization, while hydrogen bonds between invariant Y421, E352 and the glucosyl C-2 hydroxyl-group of sucrose contribute 2.15 kcal mol-1 stabilization. Changes at Y439 render predominantly hydrolytic variants synthesizing shorter oligosaccharides.
细菌蔗糖酶利用蔗糖或蔗糖类似物作为供体/受体底物来产生β(2,6)-连接的果聚糖型多糖。然而，巨大芽孢杆菌蔗糖酶（Bm-LS）的主要反应是水解。革兰氏阳性菌的单结构域蔗糖酶具有一个宽阔的底物结合口袋，且与水相通，这给构建高效转果糖基化酶带来了挑战。我们通过改变活性位点的水分布或催化酸/碱（E352）和亲核试剂（D95）的配位来实现反应特异性的转变，从而影响果糖基转移速率，并使除水以外的受体占据活性位点。对位于催化口袋第一壳层的两个丝氨酸（173/422）和两个结合水的酪氨酸（421/439）残基进行了修饰。协调D95和E3位置的S173、Y421和S422的文库变体显示出转果糖基化增加（30 - 200%）且产物谱发生改变。422位的取代对蔗糖亲和力影响更大，而173位和421位的变化对整体催化速率有强烈影响。与大多数保留型糖苷水解酶（GHs）一样，Bm-LS通过涉及两个过渡态（TS1和TS2）的双取代反应催化水解和转糖基化。D95与S173和S422侧链的氢键对TS1稳定化的贡献总计为2.4千卡/摩尔，而不变的Y421、E352与蔗糖的葡萄糖基C-2羟基之间的氢键对稳定化的贡献为2.15千卡/摩尔。Y439位的变化主要产生合成较短寡糖的水解变体。
