Teune Michelle, Vieira Plínio S, Döhler Thorben, Palm Gottfried J, Dutschei Theresa, Bartosik Daniel, Berndt Leona, Persinoti Gabriela F, Maaß Sandra, Becher Dörte, Schweder Thomas, Murakami Mário T, Lammers Michael, Bornscheuer Uwe T
Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University Greifswald, Greifswald, Germany.
Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, São Paulo, Brazil.
Nat Commun. 2025 Jul 31;16(1):7034. doi: 10.1038/s41467-025-62387-5.
Carbohydrate esterases modify polysaccharides by removing different ester moieties thereby affecting their physicochemical properties and their accessibility by glycoside hydrolases. We determined the full-length structures of two members (Fl8CE20_II and PpCE20_II) from the carbohydrate esterase family 20 (CE20) by X-ray crystallography that feature an ancillary domain, inserted into the catalytic SGNH-hydrolase domain. Detailed structural analysis identifies a so far undescribed catalytic triad architecture which lacks the typical aspartate for polarization of the histidine but instead reveals a precisely coordinated water molecule mediating contact between the His and Asp. This coordinated water in the Ser-His-(HO-Asp/Asn) motif, as further confirmed by mutational studies and by determination of kinetic constants, is crucial for catalytic activity. We therefore term this active site architecture a water-mediated catalytic triad.
碳水化合物酯酶通过去除不同的酯部分来修饰多糖，从而影响其物理化学性质以及糖苷水解酶对其的可及性。我们通过X射线晶体学确定了来自碳水化合物酯酶家族20（CE20）的两个成员（Fl8CE20_II和PpCE20_II）的全长结构，其特征是一个辅助结构域插入到催化性SGNH水解酶结构域中。详细的结构分析确定了一种迄今为止未被描述的催化三联体结构，该结构缺乏用于组氨酸极化的典型天冬氨酸，但取而代之的是揭示了一个精确配位的水分子介导组氨酸和天冬氨酸之间的接触。如通过突变研究和动力学常数测定进一步证实的那样，Ser-His-(HO-Asp/Asn)基序中的这种配位水对于催化活性至关重要。因此，我们将这种活性位点结构称为水介导的催化三联体。
