Casper Wilkens

Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77 — a mini-review

Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half of the > 45 enzymes (in 17 GH and two GT families) with an identified SBS are from GH13 and a few from GH77, both belonging to clan GH-H of carbohydrate active enzymes. The many enzymes of GH13 with SBSs provide an opportunity to analyse their distribution within this very large and diverse family. SBS containing enzymes in GH13 are spread among 15 subfamilies (two were not assigned a subfamily). Comparison of these SBSs reveals a complex evolutionary history with evidence of conservation of key residues and/or structural location between some SBSs, while others are found at entirely distinct structural locations, suggesting convergent evolution. An array of investigations of the two SBSs in barley α-amylase demonstrated they play different functional roles in binding and degradation of polysaccharides. MalQ from Escherichia coli is an α-1,4-glucanotransferase of GH77, a family that is known to have at least one member that contains an SBS. Whereas MalQ is a single domain enzyme lacking CBMs, its plant orthologue DPE2 contains two N-terminal CBM20s. Surface plasmon resonance binding studies showed that MalQ and DPE2 have a similar affinity for β-cyclodextrin and that MalQ binds malto-oligosaccharides of >DP4 at a second site in competition with β-cyclodextrin yielding a stoichiometry >1. This suggests that MalQ may have an SBS, though its structural location remains unknown.

Plant α-glucan phosphatases SEX4 and LSF2 display different affinity for amylopectin and amylose

The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for β‐cyclodextrin and validated binding site mutants deploying affinity gel electrophoresis (AGE) and surface plasmon resonance. SEX4 has a higher affinity for amylopectin; LSF2 prefers amylose and β‐cyclodextrin. SEX4 has 50‐fold lower KDapp for amylopectin compared to LSF2. Molecular dynamics simulations and AGE data both support long‐distance mutual effects of binding at SBSs and the active site in LSF2.

GH62 arabinofuranosidases: Structure, function and applications

Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify and create improved biocatalysts dedicated to plant biomass conversion. α-1,2 and α-1,3 arabinofuranosyl specific α-l-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release of α-l-arabinofuranosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plant cell walls. The CAZy database classifies α-l-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2, GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively α-l-arabinofuranosidases and these are of fungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have been characterized and very recently new knowledge was acquired with regard to crystal structures, substrate specificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62. Overall GH62 α-l-arabinofuranosidases are believed to play important roles in nature by acting in synergy with several cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnological improvements of biofuel production and in various biorefinery applications.

Selectivity of the surface binding site (SBS) on barley starch synthase I

Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable KD for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS and that the active site itself, likely working on a different end chain, has low affinity for both substrate and product.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.

Diversity of microbial carbohydrate-active enzymes in Danish anaerobic digesters fed with wastewater treatment sludge

BackgroundImproved carbohydrate-active enzymes (CAZymes) are needed to fulfill the goal of producing food, feed, fuel, chemicals, and materials from biomass. Little is known about how the diverse microbial communities in anaerobic digesters (ADs) metabolize carbohydrates or which CAZymes that are present, making the ADs a unique niche to look for CAZymes that can potentiate the enzyme blends currently used in industry.ResultsEnzymatic assays showed that functional CAZymes were secreted into the AD environments in four full-scale mesophilic Danish ADs fed with primary and surplus sludge from municipal wastewater treatment plants. Metagenomes from the ADs were mined for CAZymes with Homology to Peptide Patterns (HotPep). 19,335 CAZymes were identified of which 30% showed 50% or lower identity to known proteins demonstrating that ADs make up a promising pool for discovery of novel CAZymes. A function was assigned to 54% of all CAZymes identified by HotPep. Many different α-glucan-acting CAZymes were identified in the four metagenomes, and the most abundant family was glycoside hydrolase family 13, which contains α-glucan-acting CAZymes. Cellulytic and xylanolytic CAZymes were also abundant in the four metagenomes. The cellulytic enzymes were limited almost to endoglucanases and β-glucosidases, which reflect the large amount of partly degraded cellulose in the sludge. No dockerin domains were identified suggesting that the cellulytic enzymes in the ADs studied operate independently. Of xylanolytic CAZymes, especially xylanases and β-xylosidase, but also a battery of accessory enzymes, were present in the four ADs.ConclusionsOur findings suggest that the ADs are a good place to look for novel plant biomass degrading and modifying enzymes that can potentiate biological processes and provide basis for production of a range of added-value products from biorefineries.

Affinity Electrophoresis for Analysis of Catalytic Module-Carbohydrate Interactions

Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years, carbohydrate surface binding sites of proteins mostly enzymes have also been investigated by this method. Here, we describe a protocol for identifying binding interactions between enzyme catalytic modules and a variety of carbohydrate ligands.

Asp271 is critical for substrate interaction with the surface binding site in β-agarase A from Zobellia galactanivorans

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo‐ and exo‐modes. Agarase A (AgaA) is an endo‐glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro‐oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site‐directed mutagenesis probed by surface plasmon resonance binding analysis of agaro‐oligosaccharides of DP 4‐10. The crystal structure has shown that agaro‐octaose interacts via H‐bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA‐E147S. D271 is centrally located in the extended SBS where it forms H‐bonds to galactose and 3,6‐anhydrogalactose residues of agaro‐octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA‐E147S/D271A gave slightly decreasing KD values from 625 ± 118 to 468 ± 13 μM for agaro‐hexaose, ‐octaose, and ‐decaose, which represent 3‐ to 4‐fold reduced affinity compared with AgaA‐E147S. Molecular dynamics simulations and interaction analyses of AgaA‐E147S/D271A indicated disruption of an extended H‐bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA‐E147S/W87A nor AgaA‐E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA.

Functional roles of starch binding domains and surface binding sites in enzymes involved in starch biosynthesis

Biosynthesis of starch is catalyzed by a cascade of enzymes. The activity of a large number of these enzymes depends on interaction with polymeric substrates via carbohydrate binding sites, which are situated outside of the catalytic site and its immediate surroundings including the substrate-binding crevice. Such secondary binding sites can belong to distinct starch binding domains (SBDs), classified as carbohydrate binding modules (CBMs), or be surface binding sites (SBSs) exposed on the surface of catalytic domains. Currently in the Carbohydrate-Active enZYmes (CAZy) database SBDs are found in 13 CBM families. Four of these families; CBM20, CBM45, CBM48, and CBM53 are represented in enzymes involved in starch biosynthesis, namely starch synthases, branching enzymes, isoamylases, glucan, water dikinases, and α-glucan phosphatases. A critical role of the SBD in activity has not been demonstrated for any of these enzymes. Among the well-characterized SBDs important for starch biosynthesis are three CBM53s of Arabidopsis thaliana starch synthase III, which have modest affinity. SBSs, which are overall less widespread than SBDs, have been reported in some branching enzymes, isoamylases, synthases, phosphatases, and phosphorylases active in starch biosynthesis. SBSs appear to exert roles similar to CBMs. SBSs, however, have also been shown to modulate specificity for example by discriminating the length of chains transferred by branching enzymes. Notably, the difference in rate of occurrence between SBDs and SBSs may be due to lack of awareness of SBSs. Thus, SBSs as opposed to CBMs are not recognized at the protein sequence level, which hampers their identification. Moreover, only a few SBSs in enzymes involved in starch biosynthesis have been functionally characterized, typically by structure-guided site-directed mutagenesis. The glucan phosphatase Like SEX4 2 from A. thaliana has two SBSs with weak affinity for β-cyclodextrin, amylose and amylopectin, which were indicated by mutational analysis to be more important than the active site for initial substrate recognition. The present review provides an update on occurrence of functional SBDs and SBSs in enzymes involved in starch biosynthesis.