MOE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, People's Republic of China.
Acta Crystallogr D Struct Biol. 2021 Mar 1;77(Pt 3):336-346. doi: 10.1107/S205979832100005X. Epub 2021 Feb 17.
The products of alginate degradation, alginate oligosaccharides (AOS), have potential applications in many areas, including functional foods and marine drugs. Enzyme-based approaches using alginate lyases have advantages in the preparation of well defined AOS and have attracted much attention in recent years. However, a lack of structural insight into the whole substrate-binding cleft for most known alginate lyases severely hampers their application in the industrial generation of well defined AOS. To solve this issue, AlyF was co-crystallized with the long alginate oligosaccharide G6 (L-hexaguluronic acid hexasodium salt), which is the longest bound substrate in all solved alginate lyase complex structures. AlyF formed interactions with G6 from subsites -3 to +3 without additional substrate-binding site interactions, suggesting that the substrate-binding cleft of AlyF was fully occupied by six sugars, which was further confirmed by isothermal titration calorimetry and differential scanning calorimetry analyses. More importantly, a combination of structural comparisons and mutagenetic analyses determined that three key loops (loop 1, Lys215-Glu236; loop 2, Gln402-Ile416; loop 3, Arg334-Gly348) mainly function in binding long substrates (degree of polymerization of >4). The potential flexibility of loop 1 and loop 2 might enable the substrate to continue to enter the cleft after binding to subsites +1 to +3; loop 3 stabilizes and orients the substrate at subsites -2 and -3. Taken together, these results provide the first possible alginate lyase-substrate binding profile for long-chain alginates, facilitating the rational design of new enzymes for industrial purposes.
藻酸盐降解产物，藻酸盐寡糖（AOS），在许多领域具有潜在的应用，包括功能性食品和海洋药物。基于酶的方法使用藻酸盐裂解酶在制备具有明确定义的 AOS 方面具有优势，近年来引起了广泛关注。然而，由于大多数已知的藻酸盐裂解酶的整个底物结合裂缝缺乏结构上的了解，严重阻碍了它们在工业上生成具有明确定义的 AOS 的应用。为了解决这个问题，AlyF 与长藻酸盐寡糖 G6（L-六古洛糖醛酸六钠盐）共结晶，这是所有已解决的藻酸盐裂解酶复合物结构中最长的结合底物。AlyF 与 G6 形成了从亚位点 -3 到 +3 的相互作用，而没有额外的底物结合位点相互作用，这表明 AlyF 的底物结合裂缝完全被六个糖占据，这进一步通过等温滴定量热法和差示扫描量热法分析得到证实。更重要的是，结构比较和突变分析的结合确定了三个关键环（环 1、Lys215-Glu236；环 2、Gln402-Ile416；环 3、Arg334-Gly348）主要在结合长链底物（聚合度>4）时起作用。环 1 和环 2 的潜在灵活性可能使底物在与+1 到+3 亚位点结合后继续进入裂缝；环 3 稳定并使底物在 -2 和 -3 亚位点定向。总的来说，这些结果为长链藻酸盐提供了第一个可能的藻酸盐裂解酶-底物结合模式，为工业目的的新酶的合理设计提供了便利。
