Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract

Complete Genome Sequence of the Complex Carbohydrate-Degrading Marine Bacterium, Saccharophagus degradans Strain 2-40T

The marine bacterium Saccharophagus degradans strain 2-40 (Sde 2-40) is emerging as a vanguard of a recently discovered group of marine and estuarine bacteria that recycles complex polysaccharides. We report its complete genome sequence, analysis of which identifies an unusually large number of enzymes that degrade >10 complex polysaccharides. Not only is this an extraordinary range of catabolic capability, many of the enzymes exhibit unusual architecture including novel combinations of catalytic and substrate-binding modules. We hypothesize that many of these features are adaptations that facilitate depolymerization of complex polysaccharides in the marine environment. This is the first sequenced genome of a marine bacterium that can degrade plant cell walls, an important component of the carbon cycle that is not well-characterized in the marine environment

Carrageenan biosynthesis in red algae: A review

In this review, we summarize the current state of knowledge on the biosynthesis of carrageenan by exploring both the enzyme activities and their localizations. Genomic data, with the sequencing of the genome of Chondrus crispus and the first transcriptomic study into the life cycle stages of this organism, as well as fine carbohydrate structural determination of matrix glycans, provide leads in the study of carrageenan anabolism. Comparison to related carbohydrate-active enzymes, detailed phylogenies alongside classic histochemical studies and radioactivity assays, help predict the localization of the carrageenan-related enzyme biochemistries. Using these insights, we provide an updated model of carrageenan biosynthesis which contributes to understanding the ancestral pathway of sulfated polysaccharide biosynthesis in eukaryotes

Sweet and sour sugars from the sea: the biosynthesis and remodeling of sulfated cell wall polysaccharides from marine macroalgae

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Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens

Crystallization of a family 84 glycoside hydrolase, a putative virulence factor, secreted by C. perfringens is reported

A special issue of Essays in Biochemistry on current advances about CAZymes and their impact and key role in human health and environment

Editorial MaterialInternational audienceAbstract Carbohydrate active enzymes (CAZymes) and their biochemical characterization have been the subject of extensive research over the past ten years due to their importance to carbohydrate metabolism in different biological contexts. For instance, the understanding that ‘polysaccharide utilizing loci’ (PUL) systems hosted by specific ‘carbohydrate degraders’ in the intestinal microbiota play key roles in health and disease, such as Crohn’s disease, ulcerative colitis or colorectal cancer to name the most well-characterized, has led to an outstanding effort in trying to decipher the molecular mechanisms by which these processes are organized and regulated. The past 10 years has also seen the expansion of CAZymes with auxiliary activities, such as lytic polysaccharide monooxygenases (LPMOs) or even sulfatases, and interest has grown in general about the enzymes needed to remove the numerous decorations and modifications of complex biomass, such as carbohydrate esterases (CE). Today, the characterization of these ‘modifying’ enzymes allows us to tackle a much more complex biomass, which presents sulfations, methylations, acetylations or interconnections with lignin. This special issue about CAZyme biochemistry covers all these aspects, ranging from implications in disease to environmental and biotechnological impact, with a varied collection of twenty-four review articles providing current biochemical, structural and mechanistic insights into their respective topics

Unraveling the multivalent binding of a marine family 6 carbohydrate-binding module with its native laminarin ligand

International audienceLaminarin is an abundant brown algal storage polysaccharide. Marine microorganisms, such as Zobellia galactanivorans, produce laminarinases for its degradation, which are important for the processing of this organic matter in the ocean carbon cycle. These laminarinases are often modular, as is the case with ZgLamC which has an N‐terminal GH16 module, a central family 6 carbohydrate‐binding module (CBM) and a C‐terminal PorSS module. To date, no studies have characterized a true marine laminarin‐binding CBM6 with its natural carbohydrate ligand. The crystal structure of ZgLamCCBM6 indicates that this CBM has two clefts for binding sugar (variable loop site, VLS; and concave face site, CFS). The ZgLamCCBM6 VLS binds in an exo‐manner and the CFS interacts in an endo‐manner with laminarin. Isothermal titration calorimetry (ITC) experiments on native and mutant ZgLamCCBM6 confirm that these binding sites have different modes of recognition for laminarin, in agreement with the ‘regional model’ postulated for CBM6‐binding modules. Based on ITC data and structural data, we propose a model of ZgLamCCBM6 interacting with different chains of laminarin in a multivalent manner, forming a complex cross‐linked protein–polysaccharide network