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C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme “C-glycoside 3-oxidase” named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.

α-L-rhamnosidases play a key role in the metabolism and biodegradation of dietary flavonoid glycosides. We have developed a novel microplate spectrophotometric method to rapidly evaluate the conversion rates and substrate selectivities of mesophilic α-L-rhamnosidases towards citrus flavanone diglycosides by combining with a high-active and thermophilic β-D-glucosidase based on UV-visible spectral differences between citrus flavanone diglycosides and the corresponding aglycones under alkaline conditions. Furthermore, catalytic activities and enzyme kinetics of four α-L-rhamnosidases from human gut bacteria on various dietary flavonoid glycosides with different glycosidic bonds from various subclasses have been explored by HPLC. The α-L-rhamnosidase BtRha78A specifically removed the rhamnose group from the flavones, flavanones and flavonols diglycosides with the α-1,6 glycosidic bonds. Moreover, BtRha78A displayed higher catalytic activities on the rutinose group at 7-OH of the aglycones than at 3-OH. HFM-RhaA preferred to catalyze the flavones, flavanones and dihydrochalcones diglycosides with the α-1,2 glycosidic linkages at the 7-OH. However, this enzyme also showed high catalytic activity on the flavonol diglycoside rutin with the α-1,6 glycosidic bonds at the 3-OH. HFM-RhaC exhibited certain hydrolytic abilities towards all flavonoid diglycosides, and displayed higher activities on the flavonoid diglycosides with the α-1,6 glycosidic bonds. HFM-Rha78 weakly hydrolyzed the flavones, flavanones and dihydrochalcones diglycosides with the α-1,2 glycosidic bonds, and the flavonols diglycosides with α-1,6 glycosidic bonds. All four α-L-rhamnosidases from human gut bacteria did not exhibit catalytic activity towards the flavonoid glycosides with the α-1 glycosidic bonds. It was revealed that the α-L-rhamnosidases from human gut bacteria possessed diverse substrate selectivity on dietary flavonoid diglycosides. The structural basis for the specificity of BtRha78A on the flavonoid diglycosides with α-1,6 glycosidic bonds and the preference of HFM-RhaA on the flavonoid diglycosides with α-1,2 glycosidic bonds have been analyzed by molecular docking.

Main conclusionCarbohydrates are hydrolyzed by a family of carbohydrate-active enzymes (CAZymes) called glycosidases or glycosyl hydrolases. Here, we have summarized the roles of various plant defense glycosidases that possess different substrate specificities. We have also highlighted the open questions in this research field.Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates. Compared to those of all other sequenced organisms, plant genomes contain a remarkable diversity of glycosidases. Plant glycosidases exhibit activities on various substrates and have been shown to play important roles during pathogen infections. Plant glycosidases from different GH families have been shown to act upon pathogen components, host cell walls, host apoplastic sugars, host secondary metabolites, and host N-glycans to mediate immunity against invading pathogens. We could classify the activities of these plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. Here, we have provided comprehensive information on the catalytic activities, GH family classification, subcellular localization, domain structure, functional roles, and microbial strategies to regulate the activities of defense-related plant GHs. We have also emphasized the research gaps and potential investigations needed to advance this topic of research.

C-Glycosides, a special type of glycoside, are frequently distributed in many kinds of medicinal plants, such as puerarin and mangiferin, showing various and significant bioactivities. C-Glycosides are usually characterized by the C–C bond that forms between the anomeric carbon of sugar moieties and the carbon atom of aglycon, which is usually resistant against acidic hydrolysis and enzymatic treatments. Interestingly, C-glycosides could be cleaved by several intestinal bacteria, but whether the enzymatic cleavage of C–C glycosidic bond is reduction or hydrolysis has been controversial; furthermore, whether existence of a “C-glycosidase” directly catalyzing the cleavage is not clear. Here we review research advances about the discovery and mechanism of intestinal bacteria in enzymatic cleavage of C–C glycosidic bond with an emphasis on the identification of enzymes manipulation the deglycosylation. Finally, we give a brief conclusion about the mechanism of C-glycoside deglycosylation and perspectives for future study in this field.

Fungi secrete an array of carbohydrate-active enzymes (CAZymes), reflecting their specialized habitat-related substrate utilization. Despite its importance for fitness, enzyme secretome composition is not used in fungal classification, since an overarching relationship between CAZyme profiles and fungal phylogeny/taxonomy has not been established. For 465 Ascomycota and Basidiomycota genomes, we predicted CAZyme-secretomes, using a new peptide-based annotation method, Conserved-Unique-Peptide-Patterns, enabling functional prediction directly from sequence. We categorized each enzyme according to CAZy-family and predicted molecular function, hereby obtaining a list of “EC-Function;CAZy-Family” observations. These “Function;Family”-based secretome profiles were compared, using a Yule-dissimilarity scoring algorithm, giving equal consideration to the presence and absence of individual observations. Assessment of “Function;Family” enzyme profile relatedness (EPR) across 465 genomes partitioned Ascomycota from Basidiomycota placing Aspergillus and Penicillium among the Ascomycota. Analogously, we calculated CAZyme “Function;Family” profile-similarities among 95 Aspergillus and Penicillium species to form an alignment-free, EPR-based dendrogram. This revealed a stunning congruence between EPR categorization and phylogenetic/taxonomic grouping of the Aspergilli and Penicillia. Our analysis suggests EPR grouping of fungi to be defined both by “shared presence“ and “shared absence” of CAZyme “Function;Family” observations. This finding indicates that CAZymes-secretome evolution is an integral part of fungal speciation, supporting integration of cladogenesis and anagenesis.

α-L-Rhamnosidase can specifically hydrolyze plant natural glycosides and holds significant potential for biocatalytic applications in functional foods, healthy products, and pharmaceutical industries. Herein, a novel thermophilic and acidophilic α-L-rhamnosidase TsRha from Thermotoga sp. 2812B belonging to glycoside hydrolase family 78 was identified by genome mining and comprehensively characterized by bioinformatics, computer-aided structural analysis, and biochemical characterization. TsRha possesses a domain architecture comprising one catalytic (α/α)6-barrel domain and four β-sheet domains. TsRha displayed optimal activity at 90 °C and pH 5.0, remarkable thermostability at 80 °C, and considerable tolerance to organic solvents. TsRha exhibited broad substrate selectivity and might efficiently hydrolyze a series of natural flavonoid glycosides with various glycosidic bonds (α-1, α-1, 2, α-1, 6) from different aglycone subgroups (flavanone, flavone, flavonol, and dihydrochalcone). Moreover, it demonstrated high conversion efficiencies toward a variety of natural flavonoid diglycosides rutin, naringin, naringin dihydrochalcone, hesperidin, and troxerutin, achieving ≥99.1% conversion within 20~100 min. The excellent properties including high activity, thermophilicity, acidophilicity, good thermostability, broad substrate spectrum will make the α-L-rhamnosidase TsRha a promising biocatalyst for the efficient production of rare and high-value flavonoid glucosides with improved bioavailability and bioactivity.

Glycosylation is a critical enzymatic modification that involves the attachment of sugar moieties to target compounds, considerably influencing their physicochemical and biological characteristics. This review explored the role of two primary enzyme classes—glycosyltransferases (GTs) and glycoside hydrolases (GHs, glycosidases)—in catalyzing the glycosylation of natural products, with a specific focus on Ganoderma triterpenoids. While GTs typically use activated sugar donors, such as uridine diphosphate glucose, certain GHs can leverage more economical sugar sources, such as sucrose and starch, through transglycosylation. This paper also reviewed strategies for producing novel terpenoid glycosides, particularly recently isolated bacterial GTs and GHs capable of glycosylating terpenoids and flavonoids. It summarized the newly synthesized glycosides’ structures and biotransformation mechanisms, enhanced aqueous solubility, and potential applications. The regioselectivity and substrate specificity of GTs and GHs in catalyzing O-glycosylation (glucosylation) at distinct hydroxyl and carboxyl groups were compared. Furthermore, a special case in which the novel glycosylation reactions were mediated by GHs, including the formation of unique glycoside anomers, was included. The advantages and specific capabilities of GT/GH enzymes were evaluated for their potential in biotechnological applications and future research directions. Novel fungal triterpenoid glycosides produced through various glycosidases and sugars is expected to expand their potential applications in the future.

Xyloglucan is the most abundant hemicellulose in primary walls of spermatophytes except for grasses. Xyloglucan-degrading enzymes are important in lignocellulosic biomass hydrolysis because they remove xyloglucan, which is abundant in monocot-derived biomass. Fungal genomes encode numerous xyloglucanase genes, belonging to at least six glycoside hydrolase (GH) families. GH74 endo-xyloglucanases cleave xyloglucan backbones with unsubstituted glucose at the −1 subsite or prefer xylosyl-substituted residues in the −1 subsite. In this work, 137 GH74-related genes were detected by examining 293 Eurotiomycete genomes and Ascomycete fungi contained one or no GH74 xyloglucanase gene per genome. Another interesting feature is that the triad of tryptophan residues along the catalytic cleft was found to be widely conserved among Ascomycetes . The GH74 from Aspergillus fumigatus (AfXEG74) was chosen as an example to conduct comprehensive biochemical studies to determine the catalytic mechanism. AfXEG74 has no CBM and cleaves the xyloglucan backbone between the unsubstituted glucose and xylose-substituted glucose at specific positions, along the XX motif when linked to regions deprived of galactosyl branches. It resembles an endo-processive activity, which after initial random hydrolysis releases xyloglucan-oligosaccharides as major reaction products. This work provides insights on phylogenetic diversity and catalytic mechanism of GH74 xyloglucanases from Ascomycete fungi.

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKₐ values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKₐ values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKₐ of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

Qingzhuan tea (QZT) is a typical Chinese dark tea that has a long-time manufacturing process. In the present study, liquid chromatography coupled with tandem mass spectrometry was used to study the chemical changes of tea samples during QZT processing. Untargeted metabolomics analysis revealed that the pile-fermentation and turnover (post-fermentation, FT) was the crucial stage in transforming the main compounds of QZT, whose contents of flavan-3-ols and flavonoids glycosides were decreased significantly. The bioactivities, including the antioxidant capacities and inhibitory effects on α-amylase and α-glucosidase, were also reduced after the FT process. It was suggested that although the QZT sensory properties improved following pile-fermentation and aging, the bioactivities remained restrained. Correlation analysis indicated that the main galloylated catechins and flavonoid glycosides were highly related to their antioxidant capacity and inhibitory effects on α-amylase and α-glucosidase.