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Carbohydrate-Active enZymes (CAZymes) assemble, breakdown, and modify glycans and glycoconjugates using their catalytic and binding modules (functional protein domains). The CAZy database offers since 1998 an online and continuously updated classification of CAZyme modules (Lombard et al. 2014). Each module family in the CAZy classification has been created based on experimentally characterized protein modules from the literature, and the families are populated by related module sequences from public protein sequence databases. Since no universal threshold allows the systematic classification of the various CAZyme families, CAZy annotations result from an expert combination of module modeling/calibration and human curation. CAZy annotations are made publicly available for all proteins released by GenBank (Benson et al. 2012), Swiss-Prot (Boutet et al. 2016) and the Protein Data Bank (PDB; http://www.rcsb.org; (Berman et al. 2000)). Further, functional and 3-D structural information, curated from the literature on a regular basis, constitute essential added values to the CAZy annotation. In this spirit, the display of ligand information from crystallographic complexes has been recently developed (Lombard et al. 2014). This chapter will guide the reader through the usage of CAZy to search enzyme annotations. It will also answer frequent questions such as (i) how to obtain CAZy annotations for a specific protein, a genome, or a metagenome, (ii) how to have a newly characterized family included in the CAZy classification scheme, (iii) why CAZy does not cover all protein families related to glycans/glycoconjugates, and (iv) why CAZy does not transfer functional annotation to similar sequences. Finally, we present here a recent CAZy-associated tool, namely, the Polysaccharide Utilization Loci (PUL) predictor and database in Bacteroidetes species (Terrapon et al. 2015).

Lytic polysaccharide monooxygenases of family AA9 catalyze the oxidative cleavage of glycosidic bonds in cellulose and related polysaccharides. The N-terminal half of AA9 LPMOs displays a huge sequence variability that is in contradiction with the substrate simplicity so far observed for these enzymes. To understand the cause of the high multigenicity that prevails in the family, we have performed a clustering analysis of the N-terminal region of 3400 sequences of family AA9 LPMOs, and have evaluated the coincidence of the clusters with distal visible features that may accompany functional differences. A method based on local pairwise alignments was devised to avoid the pitfalls of a global multiple alignment. Our analysis allowed the definition of 64 clusters, which successfully segregated several visible features associated to LPMO family AA9, such as the presence of carbohydrate-binding modules, of modules of unknown function and of the conspicuous H → R substitution at the first residue of the histidine brace that holds the catalytic copper. Our analysis shows that the hypervariability of the N-terminal half of the AA9 sequences is not driven by random evolution as sequence similarity does not follow solely taxonomy. The results suggest that some clusters are perhaps able to target chitin instead of cellulose, and that preference for C1 or C4 oxidation (or lack thereof), does not appear to constitute a strong evolutionary constraint. On an evolutionary standpoint, there seems to be little constraints that apply to the N-terminal half of the sequences other than the conservation of the histidine brace. The weak evolutionary constraints that apply to the N-terminal half of AA9 LPMOs explain both their hypervariability and multigenicity.

Directeur de l'ouvrage : Arnaud Diemer Directeur de l'ouvrage : Florian Diericks Directeur de l'ouvrage : Ganna Gladkykh Directeur de l'ouvrage : Manuel E Morales Directeur de l'ouvrage : Tim Parrique Directeur de l'ouvrage : Julian Torres Auteur de l'ouvrage : Arnaud Diemer Directeur de l'ouvrage : Arnaud Diemer Directeur de l'ouvrage : Florian Diericks Directeur de l'ouvrage : Ganna Gladkykh Directeur de l'ouvrage : Manuel E Morales Directeur de l'ouvrage : Tim Parrique Directeur de l'ouvrage : Julian Torres Auteur de l'ouvrage : Arnaud DiemerDirecteur de l'ouvrage : Arnaud DiemerDirecteur de l'ouvrage : Florian DiericksDirecteur de l'ouvrage : Ganna GladkykhDirecteur de l'ouvrage : Manuel E MoralesDirecteur de l'ouvrage : Tim ParriqueDirecteur de l'ouvrage : Julian TorresAuteur de l'ouvrage : Arnaud Diemer; Exploration of fungal biodiversity for the deconstruction of lignocellulosic biomass and the implementation of new biosynthetic pathways for green chemistry. European Union and Sustainable Development: Challenges and Prospects

Since its inception, the carbohydrate-active enzymes database (CAZy; http://www.cazy.org ) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended non-catalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category. Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name “Auxiliary Activities” in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot). The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery.