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Structural insights into the substrate‐binding cleft of AlyF reveal the first long‐chain alginate‐binding mode
Author(s) -
Zhang Keke,
Liu Tao,
Liu Weizhi,
Lyu Qianqian
Publication year - 2021
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.374
H-Index - 138
ISSN - 2059-7983
DOI - 10.1107/s205979832100005x
Subject(s) - isothermal titration calorimetry , substrate (aquarium) , oligosaccharide , stereochemistry , rational design , chemistry , binding site , differential scanning calorimetry , biochemistry , combinatorial chemistry , biophysics , materials science , biology , nanotechnology , thermodynamics , ecology , physics
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.