Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII.

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation has been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Carbohydrate-binding modules enhance H2O2 tolerance by promoting lytic polysaccharide monooxygenase active site H2O2 consumption.

Lytic polysaccharide monooxygenases (LPMOs) oxidatively depolymerize recalcitrant polysaccharides, which is important for biomass conversion. The catalytic domains of many LPMOs are linked to carbohydrate-binding modules (CBMs) through flexible linkers, but the function of these CBMs in LPMO catalysis is not well understood. In this study, we utilized MtLPMO9L and MtLPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H2O2 tolerance. Using truncated forms of MtLPMO9G generated by removing the CBM, we found reduced substrate binding affinity and enzymatic activity. Conversely, when the CBM was fused to the C terminus of the single-domain MtLPMO9L to create MtLPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H2O2 tolerance. Furthermore, molecular dynamics simulations confirmed that the CBM fusion enhances the proximity of the active site to the substrate, thereby promoting multilocal cleavage and impacting the exposure of the copper active site to H2O2. Importantly, the fusion of CBM resulted in more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and reduced auto-oxidative damage of the copper active center.

Deciphering the adsorption machinery of Deep-Blue and Vp4, two myophages targeting members of the Bacillus cereus group.

In tailed phages, the baseplate is the macromolecular structure located at the tail distal part, which is directly implicated in host recognition and cell wall penetration. In myophages (i.e., with contractile tails), the baseplate is complex and comprises a central puncturing device and baseplate wedges connecting the hub to the receptor-binding proteins (RBPs). In this work, we investigated the structures and functions of adsorption-associated tail proteins of Deep-Blue and Vp4, two Herelleviridae phages infecting members of the Bacillus cereus group. Their interest resides in their different host spectrum despite a high degree of similarity. Analysis of their tail module revealed that the gene order is similar to that of the Listeria phage A511. Among their tail proteins, Gp185 (Deep-Blue) and Gp112 (Vp4) had no structural homolog, but the C-terminal variable parts of these proteins were able to bind B. cereus strains, confirming their implication in the phage adsorption. Interestingly, Vp4 and Deep-Blue adsorption to their hosts was also shown to require polysaccharides, which are likely to be bound by the arsenal of carbohydrate-binding modules (CBMs) of these phages' baseplates, suggesting that the adsorption does not rely solely on the RBPs. In particular, the BW Gp119 (Vp4), harboring a CBM fold, was shown to effectively bind to bacterial cells. Finally, we also showed that the putative baseplate hub proteins (i.e., Deep-Blue Gp189 and Vp4 Gp110) have a bacteriolytic activity against B. cereus strains, which supports their role as ectolysins locally degrading the peptidoglycan to facilitate genome injection. IMPORTANCE: The Bacillus cereus group comprises closely related species, including some with pathogenic potential (e.g., Bacillus anthracis and Bacillus cytotoxicus). Their toxins represent the most frequently reported cause of food poisoning outbreaks at the European level. Bacteriophage research is undergoing a remarkable renaissance for its potential in the biocontrol and detection of such pathogens. As the primary site of phage-bacteria interactions and a prerequisite for successful phage infection, adsorption is a crucial process that needs further investigation. The current knowledge about B. cereus phage adsorption is currently limited to siphoviruses and tectiviruses. Here, we present the first insights into the adsorption process of Herelleviridae Vp4 and Deep-Blue myophages preying on B. cereus hosts, highlighting the importance of polysaccharide moieties in this process and confirming the binding to the host surface of Deep-Blue Gp185 and Vp4 Gp112 receptor-binding proteins and Gp119 baseplate wedge.

Acoustic force spectroscopy reveals subtle differences in cellulose unbinding behavior of carbohydrate-binding modules.

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein-carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM-substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose-CBM bond rupture forces exceeding 15 pN.

Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile.

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, HhXyn5A and CtXyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both HhXyn5A and CtXyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC-PAD revealed a decreased number of reaction products for CtXyn5A with swapped CBM6, which resembled the product pattern of HhXyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity.

Exploring Host-Binding Machineries of Mycobacteriophages with AlphaFold2.

Although more than 12,000 bacteriophages infecting mycobacteria (mycobacteriophages) have been isolated so far, there is a knowledge gap on their structure-function relationships. Here, we have explored the architecture of host-binding machineries from seven representative mycobacteriophages of the Siphoviridae family infecting Mycobacterium smegmatis, Mycobacterium abscessus, and Mycobacterium tuberculosis, using AlphaFold2 (AF2). AF2 enables confident structural analyses of large and flexible biological assemblies resistant to experimental methods, thereby opening new avenues to shed light on phage structure and function. Our results highlight the modularity and structural diversity of siphophage host-binding machineries that recognize host-specific receptors at the onset of viral infection. Interestingly, the studied mycobacteriophages' host-binding machineries present unique features compared with those of phages infecting other Gram-positive actinobacteria. Although they all assemble the classical Dit (distal tail), Tal (tail-associated lysin), and receptor-binding proteins, five of them contain two potential additional adhesion proteins. Moreover, we have identified brush-like domains formed of multiple polyglycine helices which expose hydrophobic residues as potential receptor-binding domains. These polyglycine-rich domains, which have been observed in only five native proteins, may be a hallmark of mycobacteriophages' host-binding machineries, and they may be more common in nature than expected. Altogether, the unique composition of mycobacteriophages' host-binding machineries indicate they might have evolved to bind to the peculiar mycobacterial cell envelope, which is rich in polysaccharides and mycolic acids. This work provides a rational framework to efficiently produce recombinant proteins or protein domains and test their host-binding function and, hence, to shed light on molecular mechanisms used by mycobacteriophages to infect their host. IMPORTANCE Mycobacteria include both saprophytes, such as the model system Mycobacterium smegmatis, and pathogens, such as Mycobacterium tuberculosis and Mycobacterium abscessus, that are poorly responsive to antibiotic treatments and pose a global public health problem. Mycobacteriophages have been collected at a very large scale over the last decade, and they have proven to be valuable tools for mycobacteria genetic manipulation, rapid diagnostics, and infection treatment. Yet, molecular mechanisms used by mycobacteriophages to infect their host remain poorly understood. Therefore, exploring the structural diversity of mycobacteriophages' host-binding machineries is important not only to better understand viral diversity and bacteriophage-host interactions, but also to rationally develop biotechnological tools. With the powerful protein structure prediction software AlphaFold2, which was publicly released a year ago, it is now possible to gain structural and functional insights on such challenging assemblies.

Identification of chitinase from Bacillus velezensis strain S161 and its antifungal activity against Penicillium digitatum.

Previous studies have demonstrated the presence of chitinase in Bacillus velezensis through extensive genomic sequencing and experimental analyses. However, the detailed structure, functional roles, and antifungal activity of these chitinases remain poorly characterized. In this study, genomic screening identified three genes-chiA, chiB, and lpmo10-associated with chitinase degradation in B. velezensis S161. These genes encode chitinases ChiA and ChiB, and lytic polysaccharide monooxygenase LPMO10. Both ChiA and ChiB contain two CBM50 binding domains and one catalytic domain, whereas LPMO10 includes a signal peptide and a single catalytic domain. The chitinases ChiA, its truncated variant ChiA2, and ChiB were heterologously expressed in Escherichia coli. The purified enzymes efficiently degraded colloidal chitin and inhibited the spore germination of Penicillium digitatum. Notably, even after losing one CBM50 domain, the resultant enzyme, consisting of the remaining CBM50 domain and the catalytic domain, maintained its colloidal chitin hydrolysis and antifungal activity, indicating commendable stability. These results underscore the role of B. velezensis chitinases in suppressing plant pathogenic fungi and provide a solid foundation for developing and applying chitinase-based biocontrol strategies.

BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for Bacteroides starch breakdown in the human gut.

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98-CBM48-GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.

Molecular engineering of PETase for efficient PET biodegradation.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 µM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 µM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Discovery and characterization of a novel carbohydrate-binding module: a favorable tool for investigating agarose.

BACKGROUND: Agarose, mainly composed of 3,6-anhydro-α-l-galactopyranose (LA) and ß-d-galactopyranose (G) units, is an important polysaccharide with wide applications in food, biomedical and bioengineering industries. Carbohydrate-binding modules (CBMs) are favorable tools for the investigations of polysaccharides. Few agarose-binding CBMs have been hitherto reported, and their binding specificity is unclear. RESULTS: An unknown domain with a predicted ß-sandwich fold was discovered from a ß-agarase of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T . The expressed protein WfCBM101 could bind to agarose and exhibited relatively weak affinity for porphyran, with no affinity for the other seven examined polysaccharides. The protein binds to the tetrasaccharide (LA-G)2 , but not to the major tetrasaccharide contained in porphyran. The sequence novelty and well-defined binding function of WfCBM101 shed light on a novel CBM family (CBM101). Furthermore, the feasibility of WfCBM101 for visualizing agarose in situ was confirmed. CONCLUSION: A novel CBM, WfCBM101, with a desired specificity for agarose was discovered and characterized, which represents a new CBM family. The CBM could be utilized as a promising tool for studies of agarose. © 2023 Society of Chemical Industry.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure-function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal ß-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd ≤ 20 µM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6 displays activity on oat fiber xylan for prebiotic production.

Carbohydrate active enzymes are valuable tools in cereal processing to valorize underutilized side streams. By solubilizing hemicellulose and modifying the fiber structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibers: arabinoxylo-oligosaccharides. The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61°C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan. HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, whereas the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat ß-glucan. In contrast to the commercially available homolog CtXyn5A, HhXyn5A gave a more specific HPAEC-PAD oligosaccharide product profile when using wheat arabinoxylan and alkali extracted oat bran fibers as the substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modeling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX and X5 concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to the lack of important ligand-interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.

Molecular plasticity of CBM3 ancillary domain leads to conformational changes in the cellulose binding interface.

Carbohydrate-binding modules (CBMs) constitute independently folded domains typically associated with carbohydrate-active enzymes (CAZymes). These modules are considered to have a rigid structure without notable conformational changes upon ligand binding, exhibiting a complementary topography in relation to the target carbohydrate. Herein, the high-resolution SAD-solved structure of a CBM from family 3 (BsCBM3) that binds to crystalline cellulose is reported in two crystalline forms. This module showed molecular plasticity with structural differences detected between the two crystalline forms and high RMSD values when compared to NMR ensemble of models. Pronounced structural variances were observed in the cellulose binding interface between NMR and XTAL structures, which were corroborated by molecular dynamics simulations. These findings support that family 3 CBMs targeting to cellulose are rather structurally dynamic modules than rigid entities, suggesting a potential role of conformational changes in polysaccharide recognition and modulation of enzyme activity.

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts.

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nkon ) and desorption (koff ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

Distinct roles of carbohydrate-binding modules in multidomain ß-1,3-1,4-glucanase on polysaccharide degradation.

Lam16A is a novel GH16 ß-1,3-1,4-lichenase isolated from the genus Caldicellulosiruptor which can utilize untreated carbohydrate components of plant cell walls. Its catalytic module has been characterized that the six carbohydrate-binding modules (CBMs) were queued in the C-terminus, but their roles were still unclear. Here, full-length and CBM-truncated mutants of Lam16A were purified and characterized through heterologous expression in Escherichia coli. The profiles of these proteins, including the enzyme activity, degrading efficiency, substrate-binding affinity, and thermostability, were explored. Full-length Lam16A with six CBMs showed excellent thermostability and the highest activity against barley ß-glucan and laminarin with optimum pH of 6.5. The CBMs stimulated degrading ability of the catalytic module, especially against ß-1,3(4)-glucan-based polysaccharides. The released products from ß-1,3-1,4-glucan by Lam16A or its truncated mutants revealed an endo-type glycoside hydrolase. Lam16As exhibited strong binding affinities to the insoluble polysaccharides, especially Lam16A-1CBM. The degradation of yeast cell walls by Lam16A enzyme solution relative to the control reduced the absorbance values at OD800 by ~ 85% ± 1.2, enabling the release of up to ~ 0.057 ± 0.0039 µg/mL of the cytoplasmic protein into the supernatant, lowering the viability of the cells by ~ 70.3% ± 6.9, thus causing significant damage in the cell wall structure. Taken together, CBMs could influence the substrate specificity, thermal stability, and binding affinity of ß-1,3-1,4-glucanase. These results demonstrate the great potential of these enzymes to promote the bioavailability of ß-1,3-glucan oligosaccharides for health benefits. KEY POINTS: • Carbohydrate-binding modules strongly influenced the enzyme activity and binding affinity, and further impacted glycoside hydrolase activity. • Lam16A enzymes have sufficient ability to hydrolyze ß-1,3-1,4-glucan-based polysaccharides. • Lam16As provide a powerful tool to promote the bioavailability of ß-1,3-glucan oligosaccharides.

Construction of Fusion Protein with Carbohydrate-Binding Module and Leaf-Branch Compost Cutinase to Enhance the Degradation Efficiency of Polyethylene Terephthalate.

Poly(ethylene terephthalate) (PET) is a manufactured plastic broadly available, whereas improper disposal of PET waste has become a serious burden on the environment. Leaf-branch compost cutinase (LCC) is one of the most powerful and promising PET hydrolases, and its mutant LCCICCG shows high catalytic activity and excellent thermal stability. However, low binding affinity with PET has been found to dramatically limit its further industrial application. Herein, TrCBM and CfCBM were rationally selected from the CAZy database to construct fusion proteins with LCCICCG, and mechanistic studies revealed that these two domains could bind with PET favorably via polar amino acids. The optimal temperatures of LCCICCG-TrCBM and CfCBM-LCCICCG were measured to be 70 and 80 °C, respectively. Moreover, these two fusion proteins exhibited favorable thermal stability, maintaining 53.1% and 48.8% of initial activity after the incubation at 90 °C for 300 min. Compared with LCCICCG, the binding affinity of LCCICCG-TrCBM and CfCBM-LCCICCG for PET has been improved by 1.4- and 1.3-fold, respectively, and meanwhile their degradation efficiency on PET films was enhanced by 3.7% and 24.2%. Overall, this study demonstrated that the strategy of constructing fusion proteins is practical and prospective to facilitate the enzymatic PET degradation ability.

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches.

In the starch processing industry including the food and pharmaceutical industries, α-amylase is an important enzyme that hydrolyses the α-1,4 glycosidic bonds in starch, producing shorter maltooligosaccharides. In plants, starch molecules are organised in granules that are very compact and rigid. The level of starch granule rigidity affects resistance towards enzymatic hydrolysis, resulting in inefficient starch degradation by industrially available α-amylases. In an approach to enhance starch hydrolysis, the domain architecture of a Glycoside Hydrolase (GH) family 13 α-amylase from Aspergillus niger was engineered. In all fungal GH13 α-amylases that carry a carbohydrate binding domain (CBM), these modules are of the CBM20 family and are located at the C-terminus of the α-amylase domain. To explore the role of the domain order, a new GH13 gene encoding an N-terminal CBM20 domain was designed and found to be fully functional. The starch binding capacity and enzymatic activity of N-terminal CBM20 α-amylase was found to be superior to that of native GH13 without CBM20. Based on the kinetic parameters, the engineered N-terminal CBM20 variant displayed surpassing activity rates compared to the C-terminal CBM20 version for the degradation on a wide range of starches, including the more resistant raw potato starch for which it exhibits a two-fold higher Vmax underscoring the potential of domain engineering for these carbohydrate active enzymes.

Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBDGA-TuαGT and-SBDSt2-TuαGT showed Kd of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBDSt1-TuαGT had 3-5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of TuαGT fusions.

Distinct protein architectures mediate species-specific beta-glucan binding and metabolism in the human gut microbiota.

Complex glycans that evade our digestive system are major nutrients that feed the human gut microbiota (HGM). The prevalence of Bacteroidetes in the HGM of populations worldwide is engendered by the evolution of polysaccharide utilization loci (PULs), which encode concerted protein systems to utilize the myriad complex glycans in our diets. Despite their crucial roles in glycan recognition and transport, cell-surface glycan-binding proteins (SGBPs) remained understudied cogs in the PUL machinery. Here, we report the structural and biochemical characterization of a suite of SGBP-A and SGBP-B structures from three syntenic ß(1,3)-glucan utilization loci (1,3GULs) from Bacteroides thetaiotaomicron (Bt), Bacteroides uniformis (Bu), and B. fluxus (Bf), which have varying specificities for distinct ß-glucans. Ligand complexes provide definitive insight into ß(1,3)-glucan selectivity in the HGM, including structural features enabling dual ß(1,3)-glucan/mixed-linkage ß(1,3)/ß(1,4)-glucan-binding capability in some orthologs. The tertiary structural conservation of SusD-like SGBPs-A is juxtaposed with the diverse architectures and binding modes of the SGBPs-B. Specifically, the structures of the trimodular BtSGBP-B and BuSGBP-B revealed a tandem repeat of carbohydrate-binding module-like domains connected by long linkers. In contrast, BfSGBP-B comprises a bimodular architecture with a distinct ß-barrel domain at the C terminus that bears a shallow binding canyon. The molecular insights obtained here contribute to our fundamental understanding of HGM function, which in turn may inform tailored microbial intervention therapies.

Crystal structure and sugar-binding ability of the C-terminal domain of N-acetylglucosaminyltransferase IV establish a new carbohydrate-binding module family.

N-glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N-acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N-glycans in vertebrates. This enzyme attaches N-acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N-glycan core structure via a ß-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind ß-N-acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with ß-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a ß-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with ß-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of ß-N-acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability.