The IgG-specific endoglycosidases EndoS and EndoS2 are distinguished by conformation and antibody recognition.

The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodeling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific enzymes, developed to enable customizable antibody glycosylation.

Anthocyanin glucosylation mediated by a glycoside hydrolase family 3 protein in purple carrot.

Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP-sugar-dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl-glucose-dependent glucosyltransferase activity modifying cyanidin 3-xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3-type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells.

Plant N-glycan breakdown by human gut Bacteroides.

The major nutrients available to the human colonic microbiota are complex glycans derived from the diet. To degrade this highly variable mix of sugar structures, gut microbes have acquired a huge array of different carbohydrate-active enzymes (CAZymes), predominantly glycoside hydrolases, many of which have specificities that can be exploited for a range of different applications. Plant N-glycans are prevalent on proteins produced by plants and thus components of the diet, but the breakdown of these complex molecules by the gut microbiota has not been explored. Plant N-glycans are also well characterized allergens in pollen and some plant-based foods, and when plants are used in heterologous protein production for medical applications, the N-glycans present can pose a risk to therapeutic function and stability. Here we use a novel genome association approach for enzyme discovery to identify a breakdown pathway for plant complex N-glycans encoded by a gut Bacteroides species and biochemically characterize five CAZymes involved, including structures of the PNGase and GH92 α-mannosidase. These enzymes provide a toolbox for the modification of plant N-glycans for a range of potential applications. Furthermore, the keystone PNGase also has activity against insect-type N-glycans, which we discuss from the perspective of insects as a nutrient source.

A versatile fluorescence-quenched substrate for quantitative measurement of glucocerebrosidase activity within live cells.

Loss of activity of the lysosomal glycosidase ß-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.

Mechanism of 2'-fucosyllactose degradation by human-associated Akkermansia.

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2'-fucosyllactose (2'-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2'-FL. In culture, we found growth of two species, A. muciniphila MucT and A. biwaensis CSUN-19,on HMOs corresponded to a decrease in the levels of 2'-FL and an increase in lactose, indicating that the first step in 2'-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, vary between these two species. During the mid-log phase of growth, the expression of several GH29 genes was increased by 2'-FL in both species, whereas the GH95 genes were induced only in A. muciniphila. We further show that one putative fucosidase and a ß-galactosidase from A. biwaensis are involved in the breakdown of 2'-FL. Our findings indicate that the plasticity of GHs of human-associated Akkermansia sp. enables access to additional growth substrates present in HMOs, including 2'-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont.IMPORTANCEAkkermansia are mucin-degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human-associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in the growth of A. biwaensis on 2'-fucosyllactose (2'-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative ß-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2'-FL. The functional characterization of A. biwaensis growth on 2'-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR.

Enzymatic deconstruction of lignocellulosic biomass is crucial to establishment of the renewable biofuel and bioproduct economy. Better understanding of these enzymes, including their catalytic and binding domains, and other features offer potential avenues for improvement. Glycoside hydrolase family 9 (GH9) enzymes are attractive targets because they have members that exhibit exo- and endo-cellulolytic activity, processivity of reaction, and thermostability. This study examines a GH9 from Acetovibrio thermocellus ATCC 27405, AtCelR containing a catalytic domain and a carbohydrate binding module (CBM3c). Crystal structures of the enzyme without substrate, bound to cellohexaose (substrate) or cellobiose (product), show the positioning of ligands to calcium and adjacent residues in the catalytic domain that may contribute to substrate binding and facilitate product release. We also investigated the properties of the enzyme engineered to contain an additional carbohydrate binding module (CBM3a). Relative to the catalytic domain alone, CBM3a gave improved binding for Avicel (a crystalline form of cellulose), and catalytic efficiency (kcat/KM) was improved 40× with both CBM3c and CBM3a present. However, because of the molecular weight added by CBM3a, the specific activity of the engineered enzyme was not increased relative to the native construct consisting of only the catalytic and CBM3c domains. This work provides new insight into a potential role of the conserved calcium in the catalytic domain and identifies contributions and limitations of domain engineering for AtCelR and perhaps other GH9 enzymes.

Bacteroidota polysaccharide utilization system for branched dextran exopolysaccharides from lactic acid bacteria.

Dextran is an α-(1→6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with α-(1→2)-, α-(1→3)-, and α-(1→4)-linkages are often produced. Although many dextranases are known to act on the α-(1→6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of α-(1→2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade α-(1→2)- and α-(1→3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 α-glucan). The FjDexUL genes were significantly upregulated when S-32 α-glucan was the carbon source compared with α-glucooligosaccharides and α-glucans, such as linear dextran and branched α-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 α-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate α-(1→2)- and α-(1→3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on α-(1→2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of α-(1→2)- and α-(1→3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level.

GH20 and GH84 ß-N-acetylglucosaminidases with different linkage specificities underpin mucin O-glycan breakdown capability of Bifidobacterium bifidum.

Intestinal mucous layers mediate symbiosis and dysbiosis of host-microbe interactions. These interactions are influenced by the mucin O-glycan degrading ability of several gut microbes. The identities and prevalence of many glycoside hydrolases (GHs) involved in microbial mucin O-glycan breakdown have been previously reported; however, the exact mechanisms and extent to which these GHs are dedicated to mucin O-glycan degradation pathways warrant further research. Here, using Bifidobacterium bifidum as a model mucinolytic bacterium, we revealed that two ß-N-acetylglucosaminidases belonging to the GH20 (BbhI) and GH84 (BbhIV) families play important roles in mucin O-glycan degradation. Using substrate specificity analysis of natural oligosaccharides and O-glycomic analysis of porcine gastric mucin (PGM) incubated with purified enzymes or B. bifidum carrying bbhI and/or bbhIV mutations, we showed that BbhI and BbhIV are highly specific for ß-(1→3)- and ß-(1→6)-GlcNAc linkages of mucin core structures, respectively. Interestingly, we found that efficient hydrolysis of the ß-(1→3)-linkage by BbhI of the mucin core 4 structure [GlcNAcß1-3(GlcNAcß1-6)GalNAcα-O-Thr] required prior removal of the ß-(1→6)-GlcNAc linkage by BbhIV. Consistent with this, inactivation of bbhIV markedly decreased the ability of B. bifidum to release GlcNAc from PGM. When combined with a bbhI mutation, we observed that the growth of the strain on PGM was reduced. Finally, phylogenetic analysis suggests that GH84 members may have gained diversified functions through microbe-microbe and host-microbe horizontal gene transfer events. Taken together, these data strongly suggest the involvement of GH84 family members in host glycan breakdown.

Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications.

Bacteria possess diverse metabolic and genetic processes, resulting in the inability of certain bacteria to degrade trehalose. However, some bacteria do have the capability to degrade trehalose, utilizing it as a carbon source, and for defense against environmental stress. Trehalose, a disaccharide, serves as a carbon source for many bacteria, including some that are vital for pathogens. The degradation of trehalose is carried out by enzymes like trehalase (EC 3.2.1.28) and trehalose phosphorylase (EC 2.4.1.64/2.4.1.231), which are classified under the glycoside hydrolase families GH37, GH15, and GH65. Numerous studies and reports have explored the physiological functions, recombinant expression, enzymatic characteristics, and potential applications of these enzymes. However, further research is still being conducted to understand their roles in bacteria. This review aims to provide a comprehensive summary of the current understanding of trehalose degradation pathways in various bacteria, focusing on three key areas: (i) identifying different trehalose-degrading enzymes in Gram-positive and Gram-negative bacteria, (ii) elucidating the mechanisms employed by trehalose-degrading enzymes belonging to the glycoside hydrolases GH37, GH15, and GH65, and (iii) discussing the potential applications of these enzymes in different sectors. Notably, this review emphasizes the bacterial trehalose-degrading enzymes, specifically trehalases (GH37, GH15, and GH65) and trehalose phosphorylases (GH65), in both Gram-positive and Gram-negative bacteria, an aspect that has not been highlighted before.

The three-sided right-handed ß-helix is a versatile fold for glycan interactions.

Interactions between proteins and glycans are critical to various biological processes. With databases of carbohydrate-interacting proteins and increasing amounts of structural data, the three-sided right-handed ß-helix (RHBH) has emerged as a significant structural fold for glycan interactions. In this review, we provide an overview of the sequence, mechanistic, and structural features that enable the RHBH to interact with glycans. The RHBH is a prevalent fold that exists in eukaryotes, prokaryotes, and viruses associated with adhesin and carbohydrate-active enzyme (CAZyme) functions. An evolutionary trajectory analysis on structurally characterized RHBH-containing proteins shows that they likely evolved from carbohydrate-binding proteins with their carbohydrate-degrading activities evolving later. By examining three polysaccharide lyase and three glycoside hydrolase structures, we provide a detailed view of the modes of glycan binding in RHBH proteins. The 3-dimensional shape of the RHBH creates an electrostatically and spatially favorable glycan binding surface that allows for extensive hydrogen bonding interactions, leading to favorable and stable glycan binding. The RHBH is observed to be an adaptable domain capable of being modified with loop insertions and charge inversions to accommodate heterogeneous and flexible glycans and diverse reaction mechanisms. Understanding this prevalent protein fold can advance our knowledge of glycan binding in biological systems and help guide the efficient design and utilization of RHBH-containing proteins in glycobiology research.

Genetic and enzymatic characterization of Amy13E from Cellvibrio japonicus reclassifies it as a cyclodextrinase also capable of α-diglucoside degradation.

Cyclodextrinases are carbohydrate-active enzymes involved in the linearization of circular amylose oligosaccharides. Primarily thought to function as part of starch metabolism, there have been previous reports of bacterial cyclodextrinases also having additional enzymatic activities on linear malto-oligosaccharides. This substrate class also includes environmentally rare α-diglucosides such as kojibiose (α-1,2), nigerose (α-1,3), and isomaltose (α-1,6), all of which have valuable properties as prebiotics or low-glycemic index sweeteners. Previous genome sequencing of three Cellvibrio japonicus strains adapted to utilize these α-diglucosides identified multiple, but uncharacterized, mutations in each strain. One of the mutations identified was in the amy13E gene, which was annotated to encode a neopullulanase. In this report, we functionally characterized this gene and determined that it in fact encodes a cyclodextrinase with additional activities on α-diglucosides. Deletion analysis of amy13E found that this gene was essential for kojibiose and isomaltose metabolism in C. japonicus. Interestingly, a Δamy13E mutant was not deficient for cyclodextrin or pullulan utilization in C. japonicus; however, heterologous expression of the gene in E. coli was sufficient for cyclodextrin-dependent growth. Biochemical analyses found that CjAmy13E cleaved multiple substrates but preferred cyclodextrins and maltose, but had no activity on pullulan. Our characterization of the CjAmy13E cyclodextrinase is useful for refining functional enzyme predictions in related bacteria and for engineering enzymes for biotechnology or biomedical applications.IMPORTANCEUnderstanding the bacterial metabolism of cyclodextrins and rare α-diglucosides is increasingly important, as these sugars are becoming prevalent in the foods, supplements, and medicines humans consume that subsequently feed the human gut microbiome. Our analysis of a cyclomaltodextrinase with an expanded substrate range is significant because it broadens the potential applications of the GH13 family of carbohydrate active enzymes (CAZymes) in biotechnology and biomedicine. Specifically, this study provides a workflow for the discovery and characterization of novel activities in bacteria that possess a high number of CAZymes that otherwise would be missed due to complications with functional redundancy. Furthermore, this study provides a model from which predictions can be made why certain bacteria in crowded niches are able to robustly utilize rare carbon sources, possibly to gain a competitive growth advantage.

Functional studies on tandem carbohydrate-binding modules of a multimodular enzyme possessing two catalytic domains.

Although functional studies on carbohydrate-binding module (CBM) have been carried out extensively, the role of tandem CBMs in the enzyme containing multiple catalytic domains (CDs) is unclear. Here, we identified a multidomain enzyme (Lc25986) with a novel modular structure from lignocellulolytic bacterial consortium. It consists of a mannanase domain, two CBM65 domains (LcCBM65-1/LcCBM65-2), and an esterase domain. To investigate CBM function and domain interactions, full-length Lc25986 and its variants were constructed and used for enzymatic activity, binding, and bioinformatic analyses. The results showed that LcCBM65-1 and LcCBM65-2 both bind mannan and xyloglucan but not cellulose or ß-1,3-1,4-glucan, which differs from the ligand specificity of reported CBM65s. Compared to LcCBM65-2, LcCBM65-1 showed a stronger ligand affinity and a preference for acetylation sites. Both CBM65s stimulated the enzymatic activities of their respective neighboring CDs against acetylated mannan, but did not contribute to the activities of the distal CDs. The time course of mannan hydrolysis indicated that the full-length Lc25986 was more effective in the complete degradation of mixed acetyl/non-acetyl substrates than the mixture of single-CD mutants. When acting on complex substrates, LcCBM65-1 not only improved the enzymatic activity of the mannanase domain, but also directed the esterase domain to the acetylated polysaccharides. LcCBM65-2 adopted a low affinity to reduce interference with the catalysis of the mannanase domain. These results demonstrate the importance of CBMs for the synergism between the two CDs of a multidomain enzyme and suggest that they contribute to the adequate degradation of complex substrates such as plant cell walls. IMPORTANCE: Lignocellulolytic enzymes, particularly those of bacterial origin, often harbor multiple carbohydrate-binding modules (CBMs). However, the function of CBM multivalency remains poorly understood. This is especially true for enzymes that contain more than one catalytic domain (CD), as the interactions between CDs, CBMs, and CDs and CBMs can be complex. Our research demonstrates that homogeneous CBMs can have distinct functions in a multimodular enzyme. The tandem CBMs coordinate the CDs in catalytic conflict through their differences in binding affinity, ligand preference, and arrangement within the full-length enzyme. Additionally, although the synergism between mannanase and esterase is widely acknowledged, our study highlights the benefits of integrating the two enzymes into a single entity for the degradation of complex substrates. In summary, these findings enhance our understanding of the intra-synergism of a multimodular enzyme and emphasize the significance of multiple CBMs in this context.

Horizontally transferred glycoside hydrolase 26 may aid hemipteran insects in plant tissue digestion.

Glycoside hydrolases are enzymes that break down complex carbohydrates into simple sugars by catalyzing the hydrolysis of glycosidic bonds. There have been multiple instances of adaptive horizontal gene transfer of genes belonging to various glycoside hydrolase families from microbes to insects, as glycoside hydrolases can metabolize constituents of the carbohydrate-rich plant cell wall. In this study, we characterize the horizontal transfer of a gene from the glycoside hydrolase family 26 (GH26) from bacteria to insects of the order Hemiptera. Our phylogenies trace the horizontal gene transfer to the common ancestor of the superfamilies Pentatomoidea and Lygaeoidea, which include stink bugs and seed bugs. After horizontal transfer, the gene was assimilated into the insect genome as indicated by the gain of an intron, and a eukaryotic signal peptide. Subsequently, the gene has undergone independent losses and expansions in copy number in multiple lineages, suggesting an adaptive role of GH26s in some insects. Finally, we measured tissue-level gene expression of multiple stink bugs and the large milkweed bug using publicly available RNA-seq datasets. We found that the GH26 genes are highly expressed in tissues associated with plant digestion, especially in the principal salivary glands of the stink bugs. Our results are consistent with the hypothesis that this horizontally transferred GH26 was co-opted by the insect to aid in plant tissue digestion and that this HGT event was likely adaptive.

Simple Genomic Traits Predict Rates of Polysaccharide Biodegradation.

Natural and chemically modified polysaccharides are extensively employed across a wide array of industries, leading to their prevalence in the waste streams of industrialized societies. With projected increasing demand, a pressing challenge is to swiftly assess and predict their biodegradability to inform the development of new sustainable materials. In this study, we developed a scalable method to evaluate polysaccharide breakdown by measuring microbial growth and analyzing microbial genomes. Our approach, applied to polysaccharides with various structures, correlates strongly with well-established regulatory methods based on oxygen demand. We show that modifications to the polysaccharide structure decreased degradability and favored the growth of microbes adapted to break down chemically modified sugars. More broadly, we discovered two main types of microbial communities associated with different polysaccharide structures─one dominated by fast-growing microbes and another by specialized degraders. Surprisingly, we were able to predict biodegradation rates based only on two genomic features that define these communities: the abundance of genes related to rRNA (indicating fast growth) and the abundance of glycoside hydrolases (enzymes that break down polysaccharides), which together predict nearly 70% of the variation in polysaccharide breakdown. This suggests a trade-off, whereby microbes are either adapted for fast growth or for degrading complex polysaccharide chains, but not both. Finally, we observe that viral elements (prophages) encoded in the genomes of degrading microbes are induced in easily degradable polysaccharides, leading to complex dynamics in biomass accumulation during degradation. In summary, our work provides a practical approach for efficiently assessing polymer degradability and offers genomic insights into how microbes break down polysaccharides.

Development of activation-tagged gain-of-functional mutants in indica rice line (BPT 5204) for sheath blight resistance.

BACKGROUND: The development of sheath blight (ShB) resistance varieties has been a challenge for scientists for long time in rice. Activation tagging is an efficient gain-of-function mutation approach to create novel phenotypes and to identify their underlying genes. In this study, a mutant population was developed employing activation tagging in the recalcitrant indica rice (Oryza sativa L.) cv. BPT 5204 (Samba Mahsuri) through activation tagging. METHODS AND RESULTS: In this study, we have generated more than 1000 activation tagged lines in indica rice, from these mutant population 38 (GFP- RFP+) stable Ds plants were generated through germinal transposition at T2 generation based on molecular analysis and seeds selected on hygromycin (50 mg/L) containing medium segregation analyses confirmed that the transgene inherited as mendelian segregation ratio of 3:1 (3 resistant: 1 susceptible). Of them, five stable activation tagged Ds lines (M-Ds-1, M-Ds-2, M-Ds-3, M-Ds-4 and M-Ds-5) were selected based on phenotypic observation through screening for sheath blight (ShB) resistance caused by fungal pathogen Rhizoctonia solani (R. solani),. Among them, M-Ds-3 and M-Ds-5 lines showed significant resistance for ShB over other tagged lines and wild type (WT) plants. Furthermore, analysed for launch pad insertion through TAIL-PCR results and mapped on corresponding rice chromosomes. Flanking sequence and gene expression analysis revealed that the upregulation of glycoside hydrolase-OsGH or similar to Class III chitinase homologue (LOC_Os08g40680) in M-Ds-3 and a hypothetical protein gene (LOC_Os01g55000) in M-Ds-5 are potential candidate genes for sheath blight resistance in rice. CONCLUSION: In the present study, we developed Ac-Ds based ShB resistance gain-of-functional mutants through activation tagging in rice. These activation tagged mutant lines can be excellent sources for the development of ShB resistant cultivars in rice.

The genome of the invasive and broadly polyphagous Diaprepes root weevil, Diaprepes abbreviatus (Coleoptera), reveals an arsenal of putative polysaccharide-degrading enzymes.

The Diaprepes root weevil (DRW), Diaprepes abbreviatus, is a broadly polyphagous invasive pest of agriculture in the southern United States and the Caribbean. Its genome was sequenced, assembled, and annotated to study genomic correlates of specialized plant-feeding and invasiveness and to facilitate the development of new methods for DRW control. The 1.69 Gb D. abbreviatus genome assembly was distributed across 653 contigs, with an N50 of 7.8 Mb and the largest contig of 62 Mb. Most of the genome was comprised of repetitive sequences, with 66.17% in transposable elements, 5.75% in macrosatellites, and 2.06% in microsatellites. Most expected orthologous genes were present and fully assembled, with 99.5% of BUSCO genes present and 1.5% duplicated. One hundred and nine contigs (27.19 Mb) were identified as putative fragments of the X and Y sex chromosomes, and homology assessment with other beetle X chromosomes indicated a possible sex chromosome turnover event. Genome annotation identified 18,412 genes, including 43 putative horizontally transferred (HT) loci. Notably, 258 genes were identified from gene families known to encode plant cell wall degrading enzymes and invertases, including carbohydrate esterases, polysaccharide lyases, and glycoside hydrolases (GH). GH genes were unusually numerous, with 239 putative genes representing 19 GH families. Interestingly, several other beetle species with large numbers of GH genes are (like D. abbreviatus) successful invasive pests of agriculture or forestry.

Bifidobacterial GH146 ß-L-arabinofuranosidase for the removal of ß1,3-L-arabinofuranosides on plant glycans.

L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: • ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • ß-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.

Functional and structural analysis of a cyclization domain in a cyclic ß-1,2-glucan synthase.

Cyclic ß-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic ß-1,2-glucans (CßGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that ß-glucosidase-resistant compounds are produced by TiCGSCy with linear ß-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CßGs. Next, action pattern analyses using ß-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate ß-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CßGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of ß-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. KEY POINTS: • It was clearly evidenced that cyclization domain alone produces cyclic ß-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189.

Novel glycosidase from Paenibacillus lactis 154 hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins.

Oleanane-type ginsenosides are a class of compounds with remarkable pharmacological activities. However, the lack of effective preparation methods for specific rare ginsenosides has hindered the exploration of their pharmacological properties. In this study, a novel glycoside hydrolase PlGH3 was cloned from Paenibacillus lactis 154 and heterologous expressed in Escherichia coli. Sequence analysis revealed that PlGH3 consists of 749 amino acids with a molecular weight of 89.5 kDa, exhibiting the characteristic features of the glycoside hydrolase 3 family. The enzymatic characterization results of PlGH3 showed that the optimal reaction pH and temperature was 8 and 50 °C by using p-nitrophenyl-ß-D-glucopyranoside as a substrate, respectively. The Km and kcat values towards ginsenoside Ro were 79.59 ± 3.42 µM and 18.52 s-1, respectively. PlGH3 exhibits a highly specific activity on hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins. The mechanism of hydrolysis specificity was then presumably elucidated through molecular docking. Eventually, four kinds of rare oleanane-type ginsenosides (calenduloside E, pseudoginsenoside RP1, zingibroside R1, and tarasaponin VI) were successfully prepared by biotransforming total saponins extracted from Panax japonicus. This study contributes to understanding the mechanism of enzymatic hydrolysis of the GH3 family and provides a practical route for the preparation of rare oleanane-type ginsenosides through biotransformation. KEY POINTS: • The glucose at C-28 in oleanane-type saponins can be directionally hydrolyzed. • Mechanisms to interpret PlGH3 substrate specificity by molecular docking. • Case of preparation of low-sugar alternative saponins by directed hydrolysis.