Glycoside Hydrolase Family Research Articles

Crystal structure of β-d-galactofuranosidase from Streptomyces sp. JHA19 in complex with an inhibitor provides insights into substrate specificity.

d-Galactofuranose (Galf) is widely distributed in glycoconjugates of pathogenic microbes. β-d-Galactofuranosidase (Galf-ase) from Streptomyces sp. JHA19 (ORF1110) belongs to glycoside hydrolase (GH) family 2 and is the first identified Galf-specific degradation enzyme. Here, the crystal structure of ORF1110 in complex with a mechanism-based potent inhibitor, d-iminogalactitol (Ki = 65 μm) was solved. ORF1110 binds to the C5-C6 hydroxy groups of d-iminogalactitol with an extensive and integral hydrogen bond network, a key interaction that discriminates the substrates. The active site structure of ORF1110 is largely different from those of β-glucuronidases and β-galactosidases in the same GH2 family. A C-terminal domain of ORF1110 is predicted to be a carbohydrate-binding module family 42 that may bind Galf. The structural insights into Galf-ase will contribute to the investigation of therapeutic tools against pathogens.

Structural insights into the inhibition mechanism of glucosidase inhibitors toward kojibiose hydrolase belonging to the glycoside hydrolase family 65.

Glycoside hydrolase family 65 (GH65) includes glycoside hydrolases active on various α-glucosides. We previously demonstrated that the GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) is a kojibiose hydrolase and determined its three-dimensional structure. In this study, the effects of glucosidase inhibitors on FjGH65A and their complex structures were analyzed to elucidate their inhibition mechanism. FjGH65A was competitively inhibited by 1-deoxynojirimycin (DNJ) and noncompetitively inhibited by castanospermine (CSP) with Ki values of 2.95 µM and 3.69 µM, respectively. The crystal structures of FjGH65A complexed with the inhibitors indicated that DNJ was bound to subsite-1 of FjGH65A, while CSP was bound to subsites-1 and+1 of FjGH65A. Compared with the glucose-complex structure, the conformation of Tyr337 was changed in the CSP-complex structure. These results provide new structural insights into the mechanism of inhibition against GH65 α-glucoside hydrolases.

Screening, Gene Cloning and Expression of Cellulase-Producing Strain Bacillus subtilis Xh-16.

Cellulase is a complex enzyme system composed of multiple hydrolytic enzymes. It can degrade cellulose into glucose and improve the utilization efficiency of cellulose resources. Cellulase produced by microorganisms is the main method used in industry, offering the advantages of convenience and being environmentally friendly. A strain Xh-16 with high cellulase production was screened from the rotten rice straw. It was identified as Bacillus subtilis by morphological identification, physiological and biochemical identification, and 16S rRNA gene sequence analysis. Strain Xh-16 was used to degrade rice straw. After a 48h cellulase treatment, the complete degradation and structural breakdown of the straw were observed. We cloned the endoglucanase Cel5L gene of Bacillus subtilis Xh-16 and induced expression of the cloned gene in Escherichia coli BL21 (DE3). The results showed that the coding length of Cel5L gene was 1500bp, and the molecular weight of the encoded protein was about 55kDa. The molecular formula is C2456H3811N671O761S10 and it has 7709 atoms and 499 amino acids. Cel5L differs significantly from some the glycoside hydrolase family 5 cellulases because it has only one carbohydrate binding module family 3 at the C-terminus of its catalytic domain. The cellulase gene Cel5L in Xh-16 can encode active cellulase and can be heterologously expressed in Escherichia coli, which makes Escherichia coli have cellulase function. This study serves as a foundation for further research on cellulase diversity in Bacillus subtilis and offers insights for enhancing cellulase production.

Advancements in the Engineering Modification of Sucrose Phosphorylase

Sucrose phosphorylase (SPase) is a member of the glycoside hydrolase family 13, catalyzing the reversible phosphorolysis of sucrose to produce α–glucose–1–phosphate and exhibiting transglycosylation activity toward multiple substrates. Its wide substrate specificity enables the synthesis of various glycosides, which are broadly applied in food, cosmetics, and pharmaceuticals. However, the industrial application of SPase is constrained by its poor thermostability and limited transglycosylation activity. Therefore, current research focuses on enhancing the thermostability and transglycosylation activity of SPase through efficient engineering strategies based on its crystal structure and catalytic mechanism. This paper systematically reviews the crystal structure and catalytic mechanism of SPase, outlines the application of protein engineering and immobilization strategies in improving the thermostability of SPase, and analyzes how modifications at key amino acid sites affect the synthesis of typical glycosylation products. It also summarizes the limitations of SPase engineering modification strategies and explores the potential of diversified approaches for SPase modification, highlighting its broad application prospects in industrial production and laying a solid foundation for further advancements in SPase engineering modification and its industrial application.

Genome-wide identification and biochemical characterization of glycoside hydrolase gene family members in Tilletia Horrida.

Rice kernel smut, caused by Tilletia horrida, is becoming an increasingly serious disease in hybrid rice planting, leading to production losses and quality decline of male-sterile rice varieties. Successful infection requires an efficient energy source that the pathogen obtains from rice plants, such as carbohydrates. Glycoside hydrolases (GHs), one of the largest sub-families in the cell wall-degrading enzyme family, play a key role in the infection progress of pathogens. To investigate their roles in facilitating infection, in this study, we identified and characterized genes encoding GH family proteins of T. horrida and further explored the functions and structures of these genes. Through genome-wide sequencing and bioinformatics analyses, 52 GH genes were identified from T. horrida, named ThGhd_1 to ThGhd_52. The subcellular location, conserved motifs, and structures of ThGhds were identified by bioinformatics analyses. Phylogenetic analysis revealed that ThGhds with similar domains clustered together, although some proteins clustered in different branches, which might reflect functional diversity. Protein-protein interaction network analysis revealed that ThGhds interact with partner proteins involved in reactive oxygen species signaling, protein kinase activity, and plant hormone signal transduction pathways. RNA-sequencing analysis showed that the expression of ThGhd genes responded differently at different infection time points, with dynamic changes detected during the T. horrida infection process, indicating that these genes are involved in interactions with rice and have potential roles in pathogenic mechanisms. The results of this study provide valuable resources for the structure elucidation of GH family proteins of T. horrida and can help to further elucidate their roles in pathogenesis.

Degradation mechanism of difructose dianhydride III in Blautia species.

Di-fructofuranose 1,2':2,3' dianhydride (DFA-III) is a cyclic fructo-disaccharide, which is produced by the condensation of two fructose molecules via the caramelization or enzymatic reaction of inulin fructotransferase. A strain of Blautia producta was known to utilize DFA-III as a carbohydrate source; however, the mechanisms remain unclear. In this study, we characterized the glycoside hydrolase (GH) family 91 DFA-III hydrolase (DFA-IIIase) from B. parvula NBRC 113351. Recombinant BpDFA-IIIase catalyzed the reversible conversion of DFA-III to inulobiose, which is further degraded to fructose by the cooperative action of DFA-IIIase and GH32 β-D-fructofuranosidase. DFA-III was utilized in several Blautia species with a gene cluster for DFA-III degradation (e.g., B. parvula NBRC 113351, B. hydrogenotrophica JCM 14656, and B. wexlerae JCM 35486), but not by B. wexlerae JCM 31267, which does not possess the gene cluster. Furthermore, B. hansenii JCM 14655, which cannot metabolize fructose, could not utilize DFA-III; however, it could degrade DFA-III to fructose in the presence of DFA-III-degrading enzymes. Fecal fermentation tests showed that Blautia species are important gut microbe for degrading DFA-III. KEY POINTS: • BpDFA-IIIase is the first characterized DFA-IIIase in intestinal non-pathogenic bacteria. • DFA-IIIase is widely conserved in Blautia species. • DFA-III is degraded to d-fructose through inulobiose by the cooperative action of DFA-IIIase and β-d-fructofuranosidase.

Regulation of wheat yield by soil multifunctionality and metagenomic-based microbial degradation potentials under crop rotations

Crop rotation benefits soil fertility and crop yield by providing organic components including cellulose, lignin, chitin, and glucans that are mainly degraded by soil microbial carbohydrate-active enzymes (CAZymes). However, the impacts of crop rotation on soil microbial CAZyme genes are not well understood. Hence, CAZyme genes and families involved in the degradation of differentially originated organic components were investigated using metagenomics among distinct crop rotations. Crop rotation had a more significant effect on soil nitrogen than on carbon fractions with higher content in the complex rotation referring to alfalfa (Medicago sativa L.; 4 year)-potato (Solanum tuberosum L.; 1 year)-winter wheat (3 year; A4PoW3). The composition of soil microbial CAZyme genes related to the degradation of fungi-derived components was more affected by crop rotation compared with the degradation of plant- and bacteria-derived components. The total abundance of CAZyme genes and families was significantly higher in the complex rotation. Notably, CAZyme genes belonging to glycoside hydrolase and glycosyl transferase families had more connections in their network. Moreover, key genes including CE4, GH20, and GH23 assembled toward the middle of the network, and were significantly regulated by dominant soil nitrogen fractions including soil potential nitrogen mineralization and microbial biomass nitrogen. Soil multifunctionality was mostly explained by the composition and total abundance of CAZyme genes, but wheat grain yield was profoundly regulated by fungi-derived components degradation genes under effects of dominant nitrogen fractions. Overall, the findings provide deep insight into the degradation potentials of soil microbial CAZyme genes for developing sustainable crop rotational agroecosystems.

Structural analysis of gum arabic side chains from Acacia seyal released by bifidobacterial β-arabino-oligosaccharide 3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase

Gum arabic is widely used in the food and beverage industries for its emulsifying, stabilizing, and prebiotic effects, which promote Bifidobacterium growth. The two commercially approved varieties of gum arabic, namely, Acacia senegal gum and A. seyal gum, predominantly consist of arabinogalactan protein (AGP), albeit with different side chain modifications. We previously characterized two enzymes belonging to glycoside hydrolase (GH) family 39 in bifidobacteria involved in the release of α-D-Gal-(1→3)-α-L-Ara and β-L-Arap-(1→3)-α-L-Ara from the side chains of A. senegal gum. Although the carbohydrate structure of A. senegal gum is being increasingly explored, limited information is available on A. seyal gum. In this study, we discovered a novel GH39 β-arabino-oligosaccharide 3-O-β-L-arabinopyranosyl-α-L-arabinofuranosidase from Bifidobacterium catenulatum and revealed the accurate structure of β-L-arabino-oligosaccharides released from A. seyal gum as [β-L-Araf-(1→2)-]n-β-L-Arap-(1→3)-α-L-Araf-(1→) (n = 0–3). Growth assays and intracellular enzyme activity assessments using B. catenulatum revealed that β-L-arabino-oligosaccharides were degraded to L-arabinose by GH127 β-L-arabinofuranosidase and GH36 β-L-arabinopyranosidase. This study provides new insights into the diversity of AGP structures and the utilization mechanisms of A. seyal gum in bifidobacteria.

Family GH157 enzyme exhibits broad linkage tolerance and a dual endo/exo- β -glucanase activity on β-glucans

The structural and chemical diversity of β-glucans is reflected on the variety of essential biological roles tackled by these polysaccharides. This natural heterogeneity requires an elaborate assortment of enzymatic mechanisms to assemble, degrade or modify, as well as to extract their full biotechnological potential. Recent metagenomic efforts have provided an unprecedented growth in potential new biocatalysts, most of which remain unconfirmed or uncharacterized. Here we report the first biochemical and structural characterization of two bacterial β-glucanases from the recently created glycoside hydrolase family 157 (LaGH157 and BcGH157) and investigate their molecular basis for substrate hydrolysis. Structural analysis by X-ray crystallography revealed that GH157 enzymes belong to clan GH-A, possessing a (β/α)8-barrel fold catalytic domain, two β-sandwich accessory domains and two conserved catalytic glutamates residues, with relative positions compatible with a retaining mechanism of hydrolysis. Specificity screening and enzyme kinetics suggest that the enzymes prefer mixed-linkage glucans over β-1,3-glucans. Activity screening showed that both enzymes exhibit pH optimum at 6.5 and temperature optimum for LaGH157 and BcGH157 at 25 °C and 48 °C, respectively. Product analysis with HPAEC-PAD and LC-MS revealed that both enzymes are endo-1,3(4)-β-glucanases, capable of cleaving β-1,3 and β-1,4-linked glucoses, when preceded by a β-1,3 linkage. Moreover, BcGH157 needs a minimum of 4 subsites occupied for hydrolysis to occur, while LaGH157 only requires 3 subsites. Additionally, LaGH157 possesses exohydrolytic activity on β-1,3 and branching β-1,6 linkages. This unusual bifunctional endo-1,3(4)/exo-1,3–1,6 activity constitutes an expansion on our understanding of β-glucan deconstruction, with the potential to inspire future applications.

The Glucanase Enzyme Activity from Rhizospheric Streptomyces spp. in Inhibiting Growth and Damaging the Cell Walls of the Mycelium of Fusarium oxysporum

Fusarium oxysporum, a phytopathogenic fungus responsible for fusarium wilt in more than 120 plant species, is primarily managed using synthetic fungicides, which pose environmental hazards. Therefore, alternative biological control methods are urgently needed. Actinomycetes isolated from maize rhizosphere, which produce β-1.3-glucanase enzymes that degrade fungal cell wall glucans, offer promising potential as biocontrol agents. This study aimed to evaluate glucanase activity, identify genes of actinomycetes, and assess their antifungal activity against F. oxysporum. Actinomycetes demonstrating glucanase production, Streptomyces sp. ARJ 22, Streptomyces tendae ARJ 42, Streptomyces sp. ARJ 44, and Streptomyces sp. ARJ 81, were selected. Streptomyces isolates exhibited activity values ranging from 10.38 to 24.08 U/mg of protein. The presence of the bglS gene, encoding endo-β-1.3-glucanase from glycoside hydrolase family 16, supports the production of glucanase. The amino acid sequence was constructed to 3D structural model. This model exhibited high similarity to endo-β-1.3-glucanase from Nocardiopsis sp. F96. In vitro assays demonstrated that all isolates inhibited hyphal growth of F. oxysporum. Direct inhibition assays showed an average inhibition of 26.18%, whereas the filtrate culture method showed 29.38% inhibition. Enzymes from Streptomyces sp. ARJ 44 was partially purified using acetone, resulting in a specific activity of 46.34 U/mg of protein and a purity increase of up to 1.92-fold. The purified enzyme inhibited the growth of F. oxysporum mycelia by 35.80%. This inhibition was confirmed by observing damage to F. oxysporum hyphae using scanning electron microscopy. The study concluded that the four Streptomyces sp. strains producing β-1.3-glucanase enzymes have potential as biocontrol agents against F. oxysporum.

3D structural insights into the effect of N-glycosylation in human chitotriosidase variant G102S

BackgroundN-glycosylation is a key post-translational modification critical for protein function and stability. Chitotriosidase-1 (CHIT1), belonging to glycoside hydrolase family 18, is clinically utilized as a biomarker of Gaucher disease. A G102S variant is common in some populations, but the implications of this missense mutation on CHIT1 function and in disease pathology are unknown. We have investigated the effects of the G102S mutation on the N-glycosylation, structure, and activity of CHIT1. MethodsThree recombinant CHIT1 proteins, wild-type (WT), G102S, and N100Q + G102S double mutants, were expressed, purified, and analyzed for glycosylation using SDS-PAGE, MALDI-MS, PNGase F treatment, and lectin blotting. NMR and LC-MS/MS were employed to characterize glycan structures. Enzymatic assays and molecular dynamics simulations were used to assess the effects of mutations on CHIT1 function and dynamics. ResultsThe G102S mutation introduced a new N-glycosylation site at N100, confirmed by SDS-PAGE and MALDI-MS, and the composition of the N-glycan structures was verified by lectin blotting, NMR, and MS. Both G102S and N100Q + G102S proteins exhibited reduced catalytic efficiency compared to WT. Molecular dynamics simulations suggested that G102S mutation induces significant structural changes and reduces stability, particularly without N-glycan, likely impairing substrate binding and enzymatic activity. ConclusionOur findings indicate that the common G102S mutation affects the structure and function of CHIT1, partially by introducing a new N-glycosylation site. They provide a foundation for further research on the impact of N-glycosylation on its hydrolase activity and structural dynamics, with potential implications for understanding the role of CHIT1 in Gaucher disease.

Molecular Cloning, Characterization, and Application of a Novel Multifunctional Isoamylase (MIsA) from Myxococcus sp. Strain V11.

A novel multifunctional isoamylase, MIsA from Myxococcus sp. strain V11, was expressed in Escherichia coli BL21(DE3). Sequence alignment revealed that MIsA is a typical isoamylase that belongs to glycoside hydrolase family 13 (GH 13). MIsA can hydrolyze the α-1,6-branches of amylopectin and pullulan, as well as the α-1,4-glucosidic bond in amylose. Additionally, MIsA demonstrates 4-α-D-glucan transferase activity, enabling the transfer of α-1,4-glucan oligosaccharides between molecules, particularly with linear maltooligosaccharides. The Km, Kcat, and Vmax values of the MIsA for amylopectin were 1.22 mM, 40.42 µmol·min-1·mg-1, and 4046.31 mM·min-1. The yields of amylopectin and amylose hydrolyzed into oligosaccharides were 10.16% and 11.70%, respectively. The hydrolysis efficiencies were 55%, 35%, and 30% for amylopectin, soluble starch, and amylose, respectively. In the composite enzyme hydrolysis of amylose, the yield of maltotetraose increased by 1.81-fold and 2.73-fold compared with that of MIsA and MTHase (MCK8499120) alone, respectively.

Discovery of Lacto-N-Biosidases and a Novel N-Acetyllactosaminidase Activity in the CAZy Family GH20: Functional Diversity and Structural Insights.

The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.

Predicting the role of β-GAL genes in bean under abiotic stress and genome-wide characterization of β-GAL gene family members.

Β-Gals are a subgroup of the glycoside hydrolase (GH) family of enzymes, which possess the Glyco_hydro_35 (GH35) domain. Although studies have been conducted on the β-Gal gene family in numerous plant species, no such research has been conducted on beans. The purpose of this study was to determine the gene expression levels of β-Gal genes in the leaf tissue of P. vulgaris under salt and drought stress using quantitative real-time polymerase chain reaction (qRT-PCR) and to perform a comprehensive analysis of β-Gal gene family members using bioinformatics tools. In the bean genome, 25 Pvul-βGAL proteins with amino acid numbers ranging from 291 to 1119, molecular weights from 32.94 to 126.56kDa, and isoelectric points from 5.46 to 9.08 were identified. Both segmental and tandem duplication have occurred in β-Gal genes in the bean genome, and Pvul-BGAL genes have been subject to negative selection in the evolutionary process. For a deeper comprehension of the evolutionary proximity of Pvul-BGAL genes, a phylogenetic tree and synteny map were drawn together with Arabidopsis thaliana and Glycine max β-Gal genes. The expression profiles of β-Gal genes in different tissues of the bean were determined in silico. In addition, the expression profiles of β-Gal genes in the leaves of bean plants subjected to drought and salt stress were analyzed, and the role of β-Gal genes in salt and drought stress was estimated. In this study, the role of β-Gal gene family in abiotic stress response and the characterization of β-Gal genes in beans were determined for the first time and will provide a basis for future functional genomics studies.

Isolation of a novel Bacillus strain with industrial potential of producing alkaline chitosanase

A novel Bacillus strain, designated as HZ20-1, was discovered. This strain can produce naturally alkaline chitosanase without induction. Genomic analysis revealed that this chitosanase belongs to glycoside hydrolase family 8. When colloidal chitosan is used as a substrate, the maximum enzyme activity of 2.39 ± 0.03 U/mL is observed at a pH range of 8.5–9. This alkaline enzyme holds advantages over common acidic enzymes in various fields such as medicine, the environment, and daily chemical products, and thus has great market value. Moreover, as the chitosanase is a constitutive enzyme, there is no need for substrate induction. This can simplify fermentation equipment and reduce production cost. Additionally, in a preliminary study, we found that this strain can also degrade chitin and chitosan derivatives. After analysis, it was discovered that it has genes in glycoside hydrolase families 18 and 23. By controlling the enzymatic hydrolysis time, it is possible to produce products with different molecular weights, including N-acetylglucosamine, glucosamine, chito-oligosaccharides, and chitin oligosaccharides. Consequently, Bacillus sp. HZ20-1 is considered to have great potential for future industrial production of enzymes and oligosaccharides.

Genome-Wide Identification of the Maize Chitinase Gene Family and Analysis of Its Response to Biotic and Abiotic Stresses

Background/Objectives: Chitinases, enzymes belonging to the glycoside hydrolase family, play a crucial role in plant growth and stress response by hydrolyzing chitin, a natural polymer found in fungal cell walls. This study aimed to identify and analyze the maize chitinase gene family, assessing their response to various biotic and abiotic stresses to understand their potential role in plant defense mechanisms and stress tolerance. Methods: We employed bioinformatics tools to identify 43 chitinase genes in the maize B73_V5 genome. These genes were characterized for their chromosomal positions, gene and protein structures, phylogenetic relationships, functional enrichment, and collinearity. Based on previous RNA-seq data, the analysis assessed the expression patterns of these genes at different developmental stages and under multiple stress conditions. Results: The identified chitinase genes were unevenly distributed across maize chromosomes with a history of tandem duplications contributing to their divergence. The ZmChi protein family was predominantly hydrophilic and localized mainly in chloroplasts. Expression analysis revealed that certain chitinase genes were highly expressed at specific developmental stages and in response to various stresses, with ZmChi31 showing significant responsiveness to 11 different abiotic and biotic stresses. Conclusions: This study provides new insights into the role of chitinase genes in maize stress response, establishing a theoretical framework for exploring the molecular basis of maize stress tolerance. The identification of stress-responsive chitinase genes, particularly ZmChi31, offers potential candidates for further study in enhancing maize resistance to environmental challenges.

Characterization of a novel endo-1,3-fucanase from Wenyingzhuangia fucanilytica within glycoside hydrolase family 168

Sulfated fucan has attracted considerable research interest in recent years due to its diverse physiological activities. Fucanase is a critical tool for investigating sulfated fucans. In the present research, a novel endo-1,3-fucanase in the GH168 family, Fun168E, was identified within a sulfated fucan utilization loci from the genome of bacterium Wenyingzhuangia fucanilytica. Fun168E was a processive degrading enzyme and demonstrated a favorable thermostability. Ultra-performance liquid chromatography-mass spectrometry and NMR experiments demonstrated that Fun168E specifically hydrolyzed the α(1 → 3) linkages between Fucp2S and Fucp2S in sulfated fucan from Isostichopus badionotus, and α(1 → 3) linkages between Fucp2S and Fucp2,4S in sulfated fucan from Holothuria tubulosa. Fun168E could accommodate Fucp2S at subsite −1, and accept Fucp2,4S and Fucp2S at subsite +1. The discovery of this novel endo-1,3-fucanase would promote the utilization of sulfated fucans and their oligosaccharides in future applications.

QProtein: Exploring Physical Features of Protein Thermostability Based on Structural Proteomics.

Thermostability, which is essential for the functional performance of enzymes, is largely determined by intramolecular physical interactions. Although many tools have been developed, existing computational methods have struggled to find the universal principles of protein thermostability. Recent advancements in structural proteomics have been driven by the introduction of deep neural networks such as AlphaFold2 and ESMFold. These innovations have enabled the characterization of protein structures with unprecedented speed and accuracy. Here, we introduce qProtein, a Python-implemented workflow designed for the quantitative analysis of physical interactions on the scale of structural proteomics. This platform accepts protein sequences as input and produces four structural features, including hydrophobic clusters, hydrogen bonds, electrostatic interactions, and disulfide bonds. To demonstrate the use of qProtein, we investigate the structural features related to protein thermostability in six glycoside hydrolase (GH) families, comprising a total of 3,811 protein structures. Our results indicate that in five enzyme families (GH11, GH12, GH5_2, GH10, and GH48), the thermophilic enzymes have a larger average area of hydrophobic clusters compared to the nonthermophilic enzymes within each family. Furthermore, our analysis of the local-structure regions reveals that the hydrophobic clusters are predominantly distributed in the distal regions of the GH11 enzymes. In addition, the average hydrophobic cluster area of the thermophilic enzymes is significantly higher than that of the nonthermophilic enzymes in the distal regions of the GH11 enzymes. Therefore, qProtein is a well-suited platform for analyzing the structural features of thermal stability at the level of structural proteomics. We provide the source code for qProtein at https://github.com/bj600800/qProtein, and the web server is available at http://qProtein.sdu.edu.cn:8888.

Extracytoplasmic polysaccharides control cellulosomal and non-cellulosomal systems in Herbivorax saccincola A7

Herbivorax saccincola A7 is an anaerobic alkali-thermophilic lignocellulolytic bacterium that possesses a cellulosome and high xylan degradation ability. To understand the expression profile of extracellular enzymes by carbon sources, quantitative real-time PCR was performed on all cellulosomal and non-cellulosomal enzyme genes of H. saccincola A7 using cellulose and xylan as carbon sources. The results confirmed that the scaffolding proteins of H. saccincola A7 were expressed. In general, the cellulosomal genes belonging to the glycoside hydrolase families 9, 10, 11, and 48 were repressed when xylan was the sole carbon source, but these genes were significantly induced in the presence of cellulose. These results indicate that cellulose, not xylan, is a key inducer of cellulosomal genes in H. saccincola A7. The RsgI-like proteins, which regulate a carbohydrate-sensing mechanism in Clostridium thermocellum, were also found to be encoded in the H. saccincola A7 genome. To confirm the regulation by RsgI-like proteins, the relative expression of σI1–σI4 factors was analyzed on both carbon sources. The expression of alternative σI1 and σI2 factors was enhanced by the presence of cellulose. By contrast, the expression of σI3 and σI4 factors was activated by both cellulose and xylan. Taken together, the results reveal that the cellulosomal and non-cellulosomal genes of H. saccincola A7 are regulated through a carbohydrate-sensing mechanism involving anti-σ regulator RsgI-like proteins.Key points• qRT-PCR performed on cellulosomal and non-cellulosomal genes of H. saccincola A7• Cellulose is a key inducer of the cellulosome of H. saccincola A7• H. saccincola A7 possesses a similar system of anti-σ regulator RsgI-like proteins

Site-directed mutagenesis leads to the optimized transglycosylation activity of endo-beta-N-acetylglucosaminidase from Trypanosoma brucei.

Endo-β-N-acetylglucosaminidases (ENGases) are pivotal enzymes in the degradation and remodeling of glycoproteins, which catalyze the cleavage or formation of β-1,4-glycosidic bond between two N-acetylglucosamine (GlcNAc) residues in N-linked glycan chains. It was investigated that targeted mutations of amino acids in ENGases active site may modulate their hydrolytic and transglycosylation activities. Endo-Tb, the ENGase derived from Trypanosoma brucei, belongs to the glycoside hydrolase family 85 (GH85). Our group previously demonstrated that Endo-Tb exhibits hydrolytic activity toward high-mannose and complex type N-glycans and preliminarily confirmed its transglycosylation potential. In this study, we further optimized the transglycosylation activity of recombinant Endo-Tb by focusing on the N536A, E538A and Y576F mutants. A comparative analysis of their transglycosylation activity with that of the wild-type enzyme revealed that all mutants exhibited enhanced transglycosylation capacity. The N536A mutant exhibited the most pronounced improvement in transglycosylation activity with a significant reduction in hydrolytic activity. It is suggested that Endo-Tb N536A possesses the potential as a tool for synthesizing a wide array of glycoconjugates bearing high-mannose and complex type N-glycans.