Discovery of novel carbohydrate-active enzymes through the rational exploration of the protein sequences space

Abstract

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.

Structure and function of Bs164 β-mannosidase from Bacteroides salyersiae the founding member of glycoside hydrolase family GH164

“…Bs164 shows no activity towards D-xylose, D-or L-arabinose, D-galactose, Dglucose, or L-fucose containing substrates. Furthermore no activity was observed towards α-D-linked mannosides, contradicting the initial report of enzyme activity by Helbert et al (11). Bs164 did, however, have significant activity against β-linked aryl mannosides with a pH optimum 5.5 ( Figure 2).…”

Metabolism of multiple glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus

“…B. eggerthii notably outcompeted most other strains on CS-A, CS-C and HA. This could mean members of the PL35 (BACPLE_02279, BACEGG_01448) or PL37 (BACEGG_00231) which are active on GAGs 16,23 and not present in B. theta, are contributing to this enhanced growth. Fig.…”

“…Here we report how a core PUL has evolved to metabolise the complexity and heterogeneity of CS-A, CS-C, DS and HA. The PUL CS/DS/HA is coregulated with distant genes, including a hyaluronidase belonging to the recently classified PL33 16 , to complement its core activities and thus fully enabling multiple GAG breakdown. Crystal structures of the two PUL-encoded carbohydrate sulfatases provide key insights into substrate recognition by these critical, and therapeutically relevant 17 , enzymes.…”

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

“…In particular, the GH complement was suggestive of a role in ␤(1,3)-glucan hydrolysis: GH16 subfamily 3 (GH16_3 [38]) contains known endo-laminarinases (among other endo-␤-glucanases) (19) and GH3 contains exo-␤glucosidases (among others) (39). Notably, during the course of this study, GH158 emerged as a new family whose founding member was shown to hydrolyze a chemically derivatized ␤(1,3)-glucan in a high-throughput screen (40). Concordant with this proposed PUL specificity, when we probed the expression of core genes encoding the TBDT and both SGBPs (BACUNI_01487-01489), we found that they were strongly upregulated in the presence of laminarin, bMLG, and yBG as sole carbon sources versus a glucose control ( Fig.…”