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Agarose motifs, found in agars present in the cell walls of red algae, consist of alternating units of d-galactose (G) and α-3,6-anhydro-l-galactose (LA). Glycoside hydrolases from family 117 (GH117) cleave the terminal α-1,3-glycosidic bonds, releasing LA units. Structural studies have suggested that these enzymes use unconventional catalytic machinery, involving a histidine (His302) as a general acid rather than a carboxylic residue as in most glycosidases. By means of quantum mechanics/molecular mechanics metadynamics, we investigated the reaction mechanism of Phocaeicola plebeius GH117, confirming the catalytic role of His302. This residue shares a proton with a neighbor aspartate residue (Asp320), forming a His/Asp dyad. Our study also reveals that, even though the sugar unit at the -1 subsite (LA) can adopt two conformations, 4 C 1 and 1,4 B , only the latter is catalytically competent, defining a 1,4 B → [ 4 E ] ‡ → 1,4 B (→ 4 C 1 ) conformational itinerary. This mechanism may be applicable to similar enzymes with a His/Asp dyad in their active sites, such as GH3 β- N -acetylglucosaminidase and GH156 sialidase. These insights enhance our understanding of glycosidase catalytic strategies and could inform the engineering of enzymes for the more efficient processing of seaweed., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The marine Bacteroidota Zobellia galactanivorans has a polysaccharide utilization locus dedicated to the catabolism of the red algal cell wall galactan carrageenan and its unique and industrially important α-3,6-anhydro-D-galactose (ADG) monosaccharide. Here we present the first analysis of the specific molecular interactions that the exo-(α-1,3)-3,6-anhydro-D-galactosidase ZgGH129 uses to cope with the strict steric restrictions imposed by its bicyclic ADG substrate - which is ring flipped relative to D-galactose. Crystallographic snapshots of key catalytic states obtained with the natural substrate and novel chemical tools designed to mimic species along the reaction coordinate, together with quantum mechanics/molecular mechanics (QM/MM) metadynamics methods and kinetic studies, demonstrate a retaining mechanism where the second step is rate limiting. The conformational landscape of the constrained 3,6-anhydro-D-galactopyranose ring proceeds through enzyme glycosylation B 1,4 →[E 4 ] ≠ →E 4 / 1 C 4 and deglycosylation E 4 / 1 C 4 →[E 4 ] ≠ →B 1,4 itineraries limited to the Southern Hemisphere of the Cremer-Pople sphere. These results demonstrate the conformational changes throughout catalysis in a non-standard, sterically restrained, bicyclic monosaccharide, and provide a molecular framework for mechanism-based inhibitor design for anhydro-type carbohydrate-processing enzymes and for future applications involving carrageenan degradation. In addition, our study provides a rare example of distinct niche-based conformational itineraries within the same carbohydrate-active enzyme family., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Laminarin, a β(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803 T , thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota. The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization., (© 2024 The Authors. Environmental Microbiology published by John Wiley & Sons Ltd.)

Carrageenans are linear sulfated galactans synthesized in the Gigartinales, Rhodophyceae species with a varied range of biological properties that are of value to the pharmaceutical and cosmetic sectors. It is unknown how the fine structure of carrageenans dictates their capacity to affect molecular and cellular responses important to wound healing, or the ability to mitigate oxidative, hemostatic and inflammatory processes. Here we use specific endo-carrageenases, from the marine bacterium Zobellia galactanivorans, to produce enzymatically defined neo-series oligosaccharides from carrageenans with 3,6-anhydro-D-galactose on the non-reducing end. Further enzymatic modification of the oligosaccharides was done by treating with the 3,6-anhydro-D-galactosidases from the same bacterium which hydrolyze non-reducing end 3,6-anhydro-D-galactose moieties from neo-carrageenan oligosaccharides. Using the enzymatically produced oligosaccharides, we demonstrate binding to natural human serum antibodies and a monoclonal anti-αGal Ab (m86). The significant interactions with the Galα(1,3)Gal reactive antibodies produced by humans makes them potential potent inducers of complement-dependent reactions and attractive for therapeutic applications. We also demonstrate modulation of the galectin selectivity for the Gal-3 Carbohydrate Recognition Domain (CRD) relative to Gal-1 which has implications to targeting specific biological pathways regulated by the galectins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)