Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (10sup7/sup mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the α1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency ( k subcat/sub/ K subm/sub) for the synthesis of Lewis x and 3′-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix α5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.
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机译：岩藻糖基化的糖缀合物参与多种生理和病理过程。然而，岩藻糖基转移酶的较差的催化效率阻碍了岩藻糖基化药物和益生元补充剂的经济生产。在这里，我们开发了一种荧光激活的细胞分选系统，可以对此类酶进行超高通量筛选（> 10 7 突变体/小时），并设计了一种伴随策略来评估该系统的筛选性能。经过三轮定向进化，鉴定出来自幽门螺杆菌的α1,3-FucT的突变体M32，其催化效率提高了6倍和14倍（k cat / K m <分别用于合成Lewis x和3'-岩藻糖基乳糖。 M32突变体的结构表明，S45F突变产生了钳状结构，该结构似乎可以改善受体底物的吡喃半乳糖环的结合。此外，分子动力学模拟显示，螺旋线α5在M32突变体中更具移动性，可能解释了其岩藻糖基化活性高。
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