Impact of Module-X2 and Carbohydrate Binding Module-3 on the catalytic activity of associated glycoside hydrolases towards plant biomass.
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ABSTRACT:
SUBMITTER: Pasari N
PROVIDER: S-EPMC5473887 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:During the last decades, the impact of hyperthermophiles and their enzymes has been intensively investigated for implementation in various high-temperature biotechnological processes. Biocatalysts of hyperthermophiles have proven to show extremely high thermo-activities and thermo-stabilities and are identified as suitable candidates for numerous industrial processes with harsh conditions, including the process of an efficient plant biomass pretreatment and conversion. Already-characterized archaea-originated glycoside hydrolases (GHs) have shown highly impressive features and numerous enzyme characterizations indicated that these biocatalysts show maximum activities at a higher temperature range compared to bacterial ones. However, compared to bacterial biomass-degrading enzymes, the number of characterized archaeal ones remains low. To discover new promising archaeal GH candidates, it is necessary to study in detail the microbiology and enzymology of extremely high-temperature habitats, ranging from terrestrial to marine hydrothermal systems. State-of-the art technologies such as sequencing of genomes and metagenomes and automated binning of genomes out of metagenomes, combined with classical microbiological culture-dependent approaches, have been successfully performed to detect novel promising biomass-degrading hyperthermozymes. In this review, we will focus on the detection, characterization and similarities of archaeal GHs and their unique characteristics. The potential of hyperthermozymes and their impact on high-temperature industrial applications have not yet been exhausted.
| S-EPMC7469102 | biostudies-literature
Project description:Processive and distributive catalysis defines the conversion continuum, thus underpinning the transformation of oligo- and polymeric substrates by enzymes. Distributive catalysis follows an association-transformation-dissociation pattern during the formation of enzyme-reactant complexes, whereas during processive catalysis, enzymes partner with substrates and complete multiple catalytic events before dissociation from an enzyme-substrate complex. Here, we focus on processive catalysis in glycoside hydrolases (GHs), which ensures efficient conversions of substrates with high precision, and has the advantage over distributive catalysis in efficiency. The work presented here examines a recent discovery of substrate-product-assisted processive catalysis in the GH3 family enzymes with enclosed pocket-shaped active sites. We detail how GH3 β-d-glucan glucohydrolases exploit a transiently formed lateral pocket for product displacement and reactants sliding (or translocation motion) through the catalytic site without dissociation, including movements during nanoscale binding/unbinding and sliding. The phylogenetic tree of putative 550 Archaean, bacterial, fungal, Viridiplantae, and Metazoan GH3 entries resolved seven lineages that corresponded to major substrate specificity groups. This analysis indicates that two tryptophan residues in plant β-d-glucan glucohydrolases that delineate the catalytic pocket, and infer broad specificity, high catalytic efficiency, and substrate-product-assisted processivity, have evolved through a complex evolutionary process, including horizontal transfer and neo-functionalisation. We conclude that the definition of thermodynamic and mechano-structural properties of processive enzymes is fundamentally important for theoretical and practical applications in bioengineering applicable in various biotechnologies.
| S-EPMC10317168 | biostudies-literature
Project description:Most known cellulase-associated carbohydrate-binding modules (CBMs) are attached to the N- or C-terminus of the enzyme or are expressed separately and assembled into multi-enzyme complexes (for example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR-220, a GH5-family endo-β-1,4-glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A-like catalytic domain with a (β/α)8 TIM-barrel fold and an inserted CBM29-like noncatalytic domain with a β-jelly-roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full-length and CBM-deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full-length and CBM-deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single-chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross-domain groove. Subsequent site-specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase.
| S-EPMC9063844 | biostudies-literature
Project description:BackgroundDegradation via enzymatic processes for the production of valuable β-1,3-glucooligosaccharides (GOS) from curdlan has attracted considerable interest. CBM6E functions as a curdlan-specific β-1,3-endoglucanase, composed of a glycoside hydrolase family 128 (GH128) module and a carbohydrate-binding module (CBM) derived from family CBM6.ResultsCrystallographic analyses were conducted to comprehend the substrate specificity mechanism of CBM6E. This unveiled structures of both apo CBM6E and its GOS-complexed form. The GH128 and CBM6 modules constitute a cohesive unit, binding nine glucoside moieties within the catalytic groove in a singular helical conformation. By extending the substrate-binding groove, we engineered CBM6E variants with heightened hydrolytic activities, generating diverse GOS profiles from curdlan. Molecular docking, followed by mutation validation, unveiled the cooperative recognition of triple-helical β-1,3-glucan by the GH128 and CBM6 modules, along with the identification of a novel sugar-binding residue situated within the CBM6 module. Interestingly, supplementing the CBM6 module into curdlan gel disrupted the gel's network structure, enhancing the hydrolysis of curdlan by specific β-1,3-glucanases.ConclusionsThis study offers new insights into the recognition mechanism of glycoside hydrolases toward triple-helical β-1,3-glucans, presenting an effective method to enhance endoglucanase activity and manipulate its product profile. Furthermore, it discovered a CBM module capable of disrupting the quaternary structures of curdlan, thereby boosting the hydrolytic activity of curdlan gel when co-incubated with β-1,3-glucanases. These findings hold relevance for developing future enzyme and CBM cocktails useful in GOS production from curdlan degradation.
| S-EPMC10956234 | biostudies-literature
Project description:A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM) to a synthetic glycosyl hydrolase improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the CBM of the tomato (Solanum lycopersicum) SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using carboxymethylcellulose, MUC, and native crystalline cellulose assays. The presence of the CBM substantially improved the endoglucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum bicolor plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.
| S-EPMC3501001 | biostudies-literature
Project description:Chitin is an important structural component of numerous fungal pathogens and parasitic nematodes. The human macrophage chitotriosidase (HCHT) is a chitinase that hydrolyses glycosidic bonds between the N-acetyl-D-glucosamine units of this biopolymer. HCHT belongs to the Glycoside Hydrolase (GH) superfamily and contains a well-characterized catalytic domain appended to a chitin-binding domain (ChBDCHIT1). Although its precise biological function remains unclear, HCHT has been described to be involved in innate immunity. In this study, the molecular basis for interaction with insoluble chitin as well as with soluble chito-oligosaccharides has been determined. The results suggest a new mechanism as a common binding mode for many Carbohydrate Binding Modules (CBMs). Furthermore, using a phylogenetic approach, we have analysed the modularity of HCHT and investigated the evolutionary paths of its catalytic and chitin binding domains. The phylogenetic analyses indicate that the ChBDCHIT1 domain dictates the biological function of HCHT and not its appended catalytic domain. This observation may also be a general feature of GHs. Altogether, our data have led us to postulate and discuss that HCHT acts as an immune catalyser.
| S-EPMC5459812 | biostudies-literature
Project description:Four hyperthermophilic members of the bacterial genus Thermotoga (T. maritima, T. neapolitana, T. petrophila, and Thermotoga sp. strain RQ2) share a core genome of 1,470 open reading frames (ORFs), or about 75% of their genomes. Nonetheless, each species exhibited certain distinguishing features during growth on simple and complex carbohydrates that correlated with genomic inventories of specific ABC sugar transporters and glycoside hydrolases. These differences were consistent with transcriptomic analysis based on a multispecies cDNA microarray. Growth on a mixture of six pentoses and hexoses showed no significant utilization of galactose or mannose by any of the four species. T. maritima and T. neapolitana exhibited similar monosaccharide utilization profiles, with a strong preference for glucose and xylose over fructose and arabinose. Thermotoga sp. strain RQ2 also used glucose and xylose, but was the only species to utilize fructose to any extent, consistent with a phosphotransferase system (PTS) specific for this sugar encoded in its genome. T. petrophila used glucose to a significantly lesser extent than the other species. In fact, the XylR regulon was triggered by growth on glucose for T. petrophila, which was attributed to the absence of a glucose transporter (XylE2F2K2), otherwise present in the other Thermotoga species. This suggested that T. petrophila acquires glucose through the XylE1F1K1 transporter, which primarily serves to transport xylose in the other three Thermotoga species. The results here show that subtle differences exist among the hyperthermophilic Thermotogales with respect to carbohydrate utilization, which supports their designation as separate species.
| S-EPMC3298158 | biostudies-literature
Project description:The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile.
| S-EPMC5717216 | biostudies-literature
Project description:In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase (TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleaving the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2SO ring configuration as it reaches its binding site. This work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.
| S-EPMC6091422 | biostudies-literature
Project description:The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.
| S-EPMC2758015 | biostudies-literature