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.

中文翻译：

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亲核试剂的配位受损，+ 1亚位转移葡糖酶活性向果糖基化的疏水性增加

细菌糖蔗糖酶使用蔗糖或蔗糖类似物作为供体/受体底物产生β（2,6）-连接的莱万型多糖。然而，巨大芽孢杆菌的主要反应蔗糖酶（Bm-LS）是水解的。革兰氏阳性细菌的单结构域蔗糖糖酶具有宽阔的底物结合口袋，可自由接触水，这对高效反果糖基化酶的工程设计提出了挑战。我们通过改变活性位点中的水分布或催化酸/碱（E352）和亲核试剂（D95）的配位来改变反应特异性，从而影响果糖基转移速率并允许除水以外的其他受体占用活动站点。修饰位于催化袋第一壳中的两个丝氨酸（173/422）和两个与水结合的酪氨酸（421/439）残基。S173，Y421和S422的库变体与D95和E352的位置协调，显示出增加的果糖基化（30–200％）和改进的产物光谱。位置422处的取代对蔗糖亲和力具有更高的影响，而位置173和421处的改变对整体催化速率具有强烈的影响。作为大多数保留的糖苷水解酶（GHs），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的氢键与S173和S422的侧链合计贡献2.4 kcal mol^-1至TS1稳定，而不变的Y421，E352和蔗糖的葡糖基C-2羟基之间的氢键有助于2.15 kcal mol ^-1稳定。Y439的变化使水解变体主要合成较短的寡糖。