The carbohydrate-active enzyme database: functions and literature.

Thirty years have elapsed since the emergence of the classification of carbohydrate-active enzymes in sequence-based families that became the CAZy database over 20 years ago, freely available for browsing and download at www.cazy.org. In the era of large scale sequencing and high-throughput Biology, it is important to examine the position of this specialist database that is deeply rooted in human curation. The three primary tasks of the CAZy curators are (i) to maintain and update the family classification of this class of enzymes, (ii) to classify sequences newly released by GenBank and the Protein Data Bank and (iii) to capture and present functional information for each family. The CAZy website is updated once a month. Here we briefly summarize the increase in novel families and the annotations conducted during the last 8 years. We present several important changes that facilitate taxonomic navigation, and allow to download the entirety of the annotations. Most importantly we highlight the considerable amount of work that accompanies the analysis and report of biochemical data from the literature.

Trichoderma reesei Dehydrogenase, a Pyrroloquinoline Quinone-Dependent Member of Auxiliary Activity Family 12 of the Carbohydrate-Active Enzymes Database: Functional and Structural Characterization.

Pyrroloquinoline quinone (PQQ) is an ortho-quinone cofactor of several prokaryotic oxidases. Widely available in the diet and necessary for the correct growth of mice, PQQ has been suspected to be a vitamin for eukaryotes. However, no PQQ-dependent eukaryotic enzyme had been identified to use the PQQ until 2014, when a basidiomycete enzyme catalyzing saccharide dehydrogenation using PQQ as a cofactor was characterized and served to define auxiliary activity family 12 (AA12). Here we report the biochemical characterization of the AA12 enzyme encoded by the genome of the ascomycete Trichoderma reesei (TrAA12). Surprisingly, only weak activity against uncommon carbohydrates like l-fucose or d-arabinose was measured. The three-dimensional structure of TrAA12 reveals important similarities with bacterial soluble glucose dehydrogenases (sGDH). The enzymatic characterization and the structure solved in the presence of calcium confirm the importance of this ion in catalysis, as observed for sGDH. The structural characterization of TrAA12 was completed by modeling PQQ and l-fucose in the enzyme active site. Based on these results, the AA12 family of enzymes is likely to have a catalytic mechanism close to that of bacterial sGDH.IMPORTANCE Pyrroloquinoline quinone (PQQ) is an important cofactor synthesized by prokaryotes and involved in enzymatic alcohol and sugar oxidation. In eukaryotes, the benefit of PQQ as a vitamin has been suggested but never proved. Recently, the first eukaryotic enzyme using PQQ was characterized in the basidiomycete Coprinopsis cinerea, demonstrating that fungi are able to use PQQ as an enzyme cofactor. This discovery led to the classification of the fungal PQQ-dependent enzymes in auxiliary activity family 12 (AA12) of the Carbohydrate-Active Enzymes (CAZy) database (www.cazy.org) classification. In the present paper, we report on the characterization of the ascomycete AA12 enzyme from Trichoderma reesei (TrAA12). Our enzymatic and phylogenetic results show divergence with the only other member of the family characterized, that from the basidiomycete Coprinopsis cinerea The crystallographic structure of TrAA12 shows similarities to the global active-site architecture of bacterial glucose dehydrogenases, suggesting a common evolution between the two families.

Structural insights into a family 39 glycoside hydrolase from the gut symbiont Bacteroides cellulosilyticus WH2.

Bacteria from the human gut are equipped with an arsenal of carbohydrate-active enzymes that degrade dietary and host-derived glycans. In this study, we present the 2.5Å resolution crystal structure of a member (GH39wh2) from the human gut bacteria Bacteroides cellulosilyticus WH2 representative of a new subgroup within family GH39. Together with 6 other GHs, GH39wh2 belongs to a polysaccharide utilization locus (PUL) that could be involved in detecting, binding and hydrolysing a specific carbohydrate species from the intestinal tract. GH39wh2 shares a similar architecture as other members of family GH39 dominated by a typical (ß/α)8-barrel fold harboring the catalytic residues and decorated by ß-sandwich accessory domains. The GH39wh2 structure unveils an atypical shallow groove rather than a deep pocket due to drastic rearrangements in surface loops surrounding the catalytic interface. These structural adaptations seem to favour recognition of large branched substrates and may explain the lack of activity of GH39wh2 toward small xylose-based and other typical substrates from GH39 members, emphasizing the molecular diversity within the GH39 family. A phylogenetic analysis of the entire GH39 family assigns GH39wh2 as a new subgroup, consistent with the extensive remodelling of the active site region that may confer new substrate specificity toward a complex glycan chain.

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward ß-Glucans.

In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-ß(1,4)-glucanase or ß(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme fromSaccharophagus degradans(SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides.SdGluc5_26A displays lichenase activity toward ß(1,3;1,4)-glucans with a side cellobiohydrolase activity toward ß(1,4)-glucans. The three-dimensional structure ofSdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region ofSdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization ofSdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity ofSdGluc5_26A. Its deletion opens the enzyme cleft at the -3 subsite and turns the enzyme into an endo-ß(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.

Conformational flexibility of PL12 family heparinases: structure and substrate specificity of heparinase III from Bacteroides thetaiotaomicron (BT4657).

Glycosaminoglycans (GAGs) are linear polysaccharides comprised of disaccharide repeat units, a hexuronic acid, glucuronic acid or iduronic acid, linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine. GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix, control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior and differentiation. Niche adapted bacteria express enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. PL have been classified into 24 sequence-related families. Comparison of 3D structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level, and identified occurrences of convergent evolution. We have characterized structurally and enzymatically heparinase III from Bacteroides thetaiotaomicron (BtHepIII; gene BT4657), which is classified within the PL12 family. BtHepIII is a 72.5 kDa protein. We present the X-ray structures of two crystal forms of BtHepIII at resolution 1.8 and 2.4 Å. BtHepIII contains two domains, the N-terminal α-helical domain forming a toroid and the C-terminal ß-sheet domain. Comparison with recently determined structures of two other heparinases from the same PL12 family allowed us to identify structural flexibility in the arrangement of the domains indicating open-close movement. Based on comparison with other GAG lyases, we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of BtHepIII toward sulfate-poor heparan sulfate substrate.

Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate.

Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a beta-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles.

Structural snapshots of heparin depolymerization by heparin lyase I.

Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a beta-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (alpha/alpha)(6) fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.

Structural and mechanistic classification of uronic acid-containing polysaccharide lyases.

Polysaccharide lyases (PLs) have been assigned to 21 families based on their sequences, with ~ 50 singletons awaiting further classification. For 19 of these families, the structure of at least one protein is known. In this review, we have analyzed the available structural information and show that presently known PL families belong to six general folds. Only two general catalytic mechanisms have been observed among these PLs: (1) metal-assisted neutralization of the acidic group of the sugar next to the cleaved bond, with, rather unusually, arginine or lysine playing the role of Brønsted base and (2) neutralization of the acidic group on the sugar by a close approach of an amino or acidic group forcing its protonation and Tyr or Tyr-His acting as the Brønsted base and acid.

The continuing expansion of CAZymes and their families.

Carbohydrate-active enzymes (CAZymes) catalyze the assembly and breakdown of glycans and glycoconjugates. Some have been discovered, studied and exploited for numerous applications long ago. For instance, amylase and invertase were isolated in the second half of the 19th century and lysozyme was the first enzyme whose 3-D structure was determined. In spite of this early start, the number of families of carbohydrate-active enzymes continues to grow steadily in the early 21st century. This review examines the CAZyme families reported during the last two years and posits that the current expansion will continue in the future, progressively uncovering the massive diversity of glycans whose breakdown requires a large diversity of bespoke enzymes.

Improvement of the thermostability and activity of a pectate lyase by single amino acid substitutions, using a strategy based on melting-temperature-guided sequence alignment.

In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (Tm) of the enzyme more than 1 to 2 degrees C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of Xanthomonas campestris origin (PL(Xc)) produced a 6 degrees C increase in Tm and a 23-fold increase in the half-life at 45 degrees C without compromising the enzyme's catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established Tm values. Altogether, two-thirds of the nine targeted single amino acid substitutions were found to have effects either on the thermostability or on the catalytic activity of the enzyme, evidence of a high success rate of mutation without the creation of a large gene library and subsequent screening of clones. Combination of R236F with another beneficial mutation (A31G) resulted in at least a twofold increase in specific activity while preserving the improved Tm value. To understand the structural basis for the increased thermal stability or activity, the variant R236F and A31G R236F proteins and wild-type PL(Xc) were purified and crystallized. By structure analysis and computational methods, hydrophobic desolvation was found to be the driving force for the increased stability with R236F.

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.

How a Glycoside Hydrolase Recognizes a Helical Polyglucan.

Similarly to other biopolymers, linear polysaccharides can form double- or triple-helical structures. How enzymes recognize and manage this quaternary structure is an unresolved question. In this issue of Structure, Pluvinage et al. (2017) shed light on the structural complementarity between family GH81 glycoside hydrolase and the quaternary structure of their polysaccharide substrates.

Uronic polysaccharide degrading enzymes.

In the past several years progress has been made in the field of structure and function of polysaccharide lyases (PLs). The number of classified polysaccharide lyase families has increased to 23 and more detailed analysis has allowed the identification of more closely related subfamilies, leading to stronger correlation between each subfamily and a unique substrate. The number of as yet unclassified polysaccharide lyases has also increased and we expect that sequencing projects will allow many of these unclassified sequences to emerge as new families. The progress in structural analysis of PLs has led to having at least one representative structure for each of the families and for two unclassified enzymes. The newly determined structures have folds observed previously in other PL families and their catalytic mechanisms follow either metal-assisted or Tyr/His mechanisms characteristic for other PL enzymes. Comparison of PLs with glycoside hydrolases (GHs) shows several folds common to both classes but only for the ß-helix fold is there strong indication of divergent evolution from a common ancestor. Analysis of bacterial genomes identified gene clusters containing multiple polysaccharide cleaving enzymes, the Polysaccharides Utilization Loci (PULs), and their gene complement suggests that they are organized to process completely a specific polysaccharide.

Asparagine 405 of heparin lyase II prevents the cleavage of glycosidic linkages proximate to a 3-O-sulfoglucosamine residue.

Heparin and heparan sulfate contain a rare 3-O-sulfoglucosamine residue critical for anticoagulation and virus recognition, respectively. The glycosidic linkage proximate to this 3-O-sulfoglucosamine is resistant to cleavage by all heparin lyases (Heps). HepII has a broad specificity. The crystal structure of the wild type HepII identified its active site and showed a close spatial proximity between Asn405 and the 3-OH group of the bound glucosamine residue. In this study, we mutated Asn405 to the less sterically demanding Ala405 or Gly405, which broadened the substrate specificity of HepII and caused it to cleave the resistant linkage proximate to the 3-O-sulfoglucosamine residue.

Structural insights into the association between BCAR3 and Cas family members, an atypical complex implicated in anti-oestrogen resistance.

The association between novel Src homology 2-containing protein (NSP) and Crk-associated substrate (Cas) family members contributes to integrin and receptor tyrosine kinase signalling and is involved in conferring anti-oestrogen resistance to human breast carcinomas. The precise role of this association in tumorigenesis remains controversial, and the molecular basis for the complex NSP and Cas protein form is unknown. Here we present a pluridisciplinary approach, including small-angle X-ray scattering, that provides first insights into the structure of the complex formed between breast cancer anti-oestrogen resistance 3 (BCAR3, an NSP family member) and human enhancer of filamentation 1 (HEF1, also named NEDD9 or Cas-L, a Cas family protein). Our analysis corroborates a four-helix bundle structure for the NSP-binding domain of HEF1 and a Cdc25-like guanine nucleotide exchange factor (GEF) fold for the Cas-binding domain of BCAR3. Using residues located on helix 2 of the four-helix bundle, HEF1 binds very tightly to a site on BCAR3 that is remote from the putative guanosine triphosphatase binding site of the GEF domain, but similar to a site implicated in allosteric regulation of the homologous SOS (Son of Sevenless) GEF domain. Thus, the association between NSP and Cas proteins might not only create a very stable link between these molecules, co-localising their cellular functions, but also modulate the function of the NSP GEF domains. Such modulation may explain, at least in part, the controversial results published for NSP GEF function.

Structural basis for the interaction between focal adhesion kinase and CD4.

Focal adhesion kinase (FAK) and CD4 fulfil vital functions in cellular signal transduction: FAK is a central component in integrin signalling, whereas CD4 plays essential roles in the immune defence. In T lymphocytes, FAK and CD4 localise to the same signalling complexes after stimulation by either the human immunodeficiency virus (HIV) gp120 glycoprotein or an antigen, suggesting the concerted action of FAK and CD4 in these cells. Using crystallography and microcalorimetry, we here show that the focal adhesion targeting (FAT) domain of FAK binds specifically to the CD4 endocytosis motif in vitro. This FAT-CD4 complex is structurally and thermodynamically similar to the one FAT forms with paxillin LD motifs. The CD4 binding site on FAT presents the same features as the established CD4 binding site on the HIV-1 Nef protein. The binding of FAT to CD4 is incompatible with the binding of Lck to CD4. We further show that HIV-1 gp120 triggers the association of CD4 with FAK in T cells, under conditions that are known to dissociate Lck from CD4. Our results suggest that the FAK-CD4 complex represents an alternative route for eliciting T-cell-specific signals and that it links gp120 engagement to distinctive T-cell signalling during HIV infection. In infected cells, HIV-1 Nef may displace FAK from CD4 to protect the cells from apoptosis.