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1.
Carbohydr Polym ; 341: 122297, 2024 Oct 01.
Article in English | MEDLINE | ID: mdl-38876729

ABSTRACT

The biological activities of heparan sulfate (HS) are intimately related to their molecular weights, degree and pattern of sulfation and homogeneity. The existing methods for synthesizing homogeneous sugar chains of low dispersity involve multiple steps and require stepwise isolation and purification processes. Here, we designed a mesoporous metal-organic capsule for the encapsulation of glycosyltransferase and obtained a microreactor capable of enzymatically catalyzing polymerization reactions to prepare homogeneous heparosan of low dispersity, the precursor of HS and heparin. Since the sugar chain extension occurs in the pores of the microreactor, low molecular weight heparosan can be synthesized through space-restricted catalysis. Moreover, the glycosylation co-product, uridine diphosphate (UDP), can be chelated with the exposed metal sites of the metal-organic capsule, which inhibits trans-cleavage to reduce the molecular weight dispersity. This microreactor offers the advantages of efficiency, reusability, and obviates the need for stepwise isolation and purification processes. Using the synthesized heparosan, we further successfully prepared homogeneous 6-O-sulfated HS of low dispersity with a molecular weight of approximately 6 kDa and a polydispersity index (PDI) of 1.032. Notably, the HS generated exhibited minimal anticoagulant activity, and its binding affinity to fibroblast growth factor 1 was comparable to that of low molecular weight heparins.


Subject(s)
Heparitin Sulfate , Polymerization , Heparitin Sulfate/chemistry , Anticoagulants/chemistry , Anticoagulants/pharmacology , Anticoagulants/chemical synthesis , Molecular Weight , Porosity , Humans , Disaccharides/chemistry , Glycosyltransferases/metabolism , Glycosyltransferases/chemistry
2.
Protein Sci ; 33(7): e5038, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38864725

ABSTRACT

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.


Subject(s)
Bacterial Proteins , Pseudomonas aeruginosa , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Periplasm/metabolism , Periplasm/enzymology , Periplasmic Proteins/metabolism , Periplasmic Proteins/genetics , Periplasmic Proteins/chemistry , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/chemistry , Peptidoglycan/metabolism , Peptidoglycan/chemistry
3.
Plant Signal Behav ; 19(1): 2360296, 2024 Dec 31.
Article in English | MEDLINE | ID: mdl-38808631

ABSTRACT

Rainfall, wind and touch, as mechanical forces, were mimicked on 6-week-old soil-grown tomato and potato under controlled conditions. Expression level changes of xyloglucan endotransglucosylase/hydrolase genes (XTHs) of tomato (Solanum lycopersicum L. cv. Micro Tom; SlXTHs) and potato (Solanum tuberosum L. cv. Desirée; StXTHs) were analyzed in response to these mechanical forces. Transcription intensity of every SlXTHs of tomato was altered in response to rainfall, while the expression intensity of 72% and 64% of SlXTHs was modified by wind and touch, respectively. Ninety-one percent of StXTHs (32 out of 35) in potato responded to the rainfall, while 49% and 66% of the StXTHs were responsive to the wind and touch treatments, respectively. As previously demonstrated, all StXTHs were responsive to ultrasound treatment, and all were sensitive to one or more of the environmental mechanical factors examined in the current study. To our best knowledge, this is the first study to demonstrate that these ubiquitous mechanical environmental cues, such as rainfall, wind and touch, influence the transcription of most XTHs examined in both species.


Subject(s)
Gene Expression Regulation, Plant , Rain , Solanum lycopersicum , Solanum tuberosum , Wind , Solanum lycopersicum/genetics , Solanum lycopersicum/metabolism , Solanum tuberosum/genetics , Solanum tuberosum/metabolism , Solanum tuberosum/physiology , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Touch/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Genes, Plant
4.
Biomacromolecules ; 25(6): 3807-3822, 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38807305

ABSTRACT

Glycans, composed of linked monosaccharides, play crucial roles in biology and find diverse applications. Enhancing their enzymatic synthesis can be achieved by immobilizing enzymes on materials such as microgels. Here, we present microgels with immobilized glycosyltransferases, synthesized through droplet microfluidics, immobilizing enzymes either via encapsulation or postattachment. SpyTag-SpyCatcher interaction was used for enzyme binding, among others. Fluorescamine and permeability assays confirmed enzyme immobilization and microgel porosity, while enzymatic activities were determined using HPLC. The potential application of microgels in cascade reactions involving multiple enzymes was demonstrated by combining ß4GalT and α3GalT in an enzymatic reaction with high yields. Moreover, a cascade of ß4GalT and ß3GlcNAcT was successfully implemented. These results pave the way toward a modular membrane bioreactor for automated glycan synthesis containing the presented biocatalytic microgels.


Subject(s)
Enzymes, Immobilized , Glycosyltransferases , Microgels , Polysaccharides , Enzymes, Immobilized/chemistry , Polysaccharides/chemistry , Glycosyltransferases/metabolism , Glycosyltransferases/chemistry , Microgels/chemistry , Biocatalysis
5.
Nat Commun ; 15(1): 4588, 2024 May 30.
Article in English | MEDLINE | ID: mdl-38816433

ABSTRACT

Lycibarbarspermidines are unusual phenolamide glycosides characterized by a dicaffeoylspermidine core with multiple glycosyl substitutions, and serve as a major class of bioactive ingredients in the wolfberry. So far, little is known about the enzymatic basis of the glycosylation of phenolamides including dicaffeoylspermidine. Here, we identify five lycibarbarspermidine glycosyltransferases, LbUGT1-5, which are the first phenolamide-type glycosyltransferases and catalyze regioselective glycosylation of dicaffeoylspermidines to form structurally diverse lycibarbarspermidines in wolfberry. Notably, LbUGT3 acts as a distinctive enzyme that catalyzes a tandem sugar transfer to the ortho-dihydroxy group on the caffeoyl moiety to form the unusual ortho-diglucosylated product, while LbUGT1 accurately discriminates caffeoyl and dihydrocaffeoyl groups to catalyze a site-selective sugar transfer. Crystal structure analysis of the complexes of LbUGT1 and LbUGT3 with UDP, combined with molecular dynamics simulations, revealed the structural basis of the difference in glycosylation selectivity between LbUGT1 and LbUGT3. Site-directed mutagenesis illuminates a conserved tyrosine residue (Y389 in LbUGT1 and Y390 in LbUGT3) in PSPG box that plays a crucial role in regulating the regioselectivity of LbUGT1 and LbUGT3. Our study thus sheds light on the enzymatic underpinnings of the chemical diversity of lycibarbarspermidines in wolfberry, and expands the repertoire of glycosyltransferases in nature.


Subject(s)
Glycosyltransferases , Lycium , Glycosyltransferases/metabolism , Glycosyltransferases/chemistry , Glycosyltransferases/genetics , Glycosylation , Lycium/enzymology , Lycium/metabolism , Lycium/chemistry , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Plant Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/chemistry , Glycosides/metabolism , Glycosides/chemistry , Crystallography, X-Ray , Piperidines/metabolism , Piperidines/chemistry , Substrate Specificity
6.
Food Funct ; 15(11): 6042-6053, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38752441

ABSTRACT

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin produced by Fusarium spp., contaminates cereals and threatens human and animal health by inducing hepatotoxicity, immunotoxicity, and genotoxicity. In this study, a new Bacillus subtilis strain, YQ-1, with a strong ability to detoxify ZEN, was isolated from soil samples and characterized. YQ-1 was confirmed to degrade more than 46.26% of 20 µg mL-1 ZEN in Luria-Bertani broth and 98.36% in fermentation broth within 16 h at 37 °C; one of the two resulting products was ZEN-diglucoside. Under optimal reaction conditions (50 °C and pH 5.0-9.0), the reaction mixture generated by YQ-1 catalyzing ZEN significantly reduced the promoting effect of ZEN on MCF-7 cell proliferation, effectively eliminating the estrogenic toxicity of ZEN. In addition, a new glycosyltransferase gene (yqgt) from B. subtilis YQ-1 was cloned with 98% similarity to Bs-YjiC from B. subtilis 168 and over-expressed in E. coli BL21 (DE3). ZEN glycosylation activity converted 25.63% of ZEN (20 µg mL-1) to ZEN-diG after 48 h of reaction at 37 °C. The characterization of ZEN degradation by B. subtilis YQ-1 and the expression of YQGT provide a theoretical basis for analyzing the mechanism by which Bacillus spp. degrades ZEN.


Subject(s)
Bacillus subtilis , Glycosyltransferases , Zearalenone , Zearalenone/metabolism , Zearalenone/chemistry , Bacillus subtilis/metabolism , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Humans , Glycosylation , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism
7.
J Agric Food Chem ; 72(23): 13328-13340, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38805380

ABSTRACT

Flavonol glycosides, contributing to the health benefits and distinctive flavors of tea (Camellia sinensis), accumulate predominantly as diglycosides and triglycosides in tea leaves. However, the UDP-glycosyltransferases (UGTs) mediating flavonol multiglycosylation remain largely uncharacterized. In this study, we employed an integrated proteomic and metabolomic strategy to identify and characterize key UGTs involved in flavonol triglycoside biosynthesis. The recombinant rCsUGT75AJ1 exhibited flavonoid 4'-O-glucosyltransferase activity, while rCsUGT75L72 preferentially catalyzed 3-OH glucosylation. Notably, rCsUGT73AC15 displayed substrate promiscuity and regioselectivity, enabling glucosylation of rutin at multiple sites and kaempferol 3-O-rutinoside (K3R) at the 7-OH position. Kinetic analysis revealed rCsUGT73AC15's high affinity for rutin (Km = 9.64 µM). Across cultivars, CsUGT73AC15 expression inversely correlated with rutin levels. Moreover, transient CsUGT73AC15 silencing increased rutin and K3R accumulation while decreasing their respective triglycosides in tea plants. This study offers new mechanistic insights into the key roles of UGTs in regulating flavonol triglycosylation in tea plants.


Subject(s)
Camellia sinensis , Flavonols , Glycosides , Glycosyltransferases , Plant Proteins , Camellia sinensis/genetics , Camellia sinensis/metabolism , Camellia sinensis/enzymology , Camellia sinensis/chemistry , Plant Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/chemistry , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/chemistry , Flavonols/metabolism , Flavonols/chemistry , Flavonols/biosynthesis , Glycosides/metabolism , Glycosides/chemistry , Plant Leaves/metabolism , Plant Leaves/chemistry , Plant Leaves/genetics , Plant Leaves/enzymology , Kinetics , Rutin/metabolism , Rutin/chemistry
8.
Int J Biol Macromol ; 270(Pt 2): 132228, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38734355

ABSTRACT

Panonychus citri (McGregor) strains have developed a high level of resistance to abamectin, but the underlying molecular mechanism is unknown. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are critical for the removal of a variety of exogenous and endogenous substances. In this study, an enzyme activity assay revealed that UGTs potentially contribute to P. citri abamectin resistance. Spatiotemporal expression profiles showed that only PcUGT202A9 was significantly overexpressed in the abamectin-resistant strain (AbR) at all developmental stages. Moreover, UGT activity decreased significantly, whereas abamectin susceptibility increased significantly, in AbR after PcUGT202A9 was silenced. Three-dimensional modeling and molecular docking analyses revealed that PcUGT202A9 can bind stably to abamectin. Recombinant PcUGT202A9 activity was detected when α-naphthol was used, but the enzymatic activity was inhibited by abamectin (50 % inhibitory concentration: 803.3 ±â€¯14.20 µmol/L). High-performance liquid chromatography and mass spectrometry analyses indicated that recombinant PcUGT202A9 can effectively degrade abamectin and catalyze the conjugation of UDP-glucose to abamectin. These results imply PcUGT202A9 contributes to P. citri abamectin resistance.


Subject(s)
Glycosyltransferases , Ivermectin , Molecular Docking Simulation , Ivermectin/analogs & derivatives , Ivermectin/pharmacology , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Glycosyltransferases/chemistry , Animals , Drug Resistance/genetics
9.
Proc Natl Acad Sci U S A ; 121(21): e2402554121, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38748580

ABSTRACT

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.


Subject(s)
Glycosyltransferases , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Glycosylation , Citrobacter/metabolism , Citrobacter/genetics , O Antigens/metabolism , O Antigens/chemistry , Polysaccharides/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Polysaccharides, Bacterial/metabolism
10.
Appl Environ Microbiol ; 90(6): e0220323, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38747588

ABSTRACT

The O antigen (OAg) polysaccharide is one of the most diverse surface molecules of Gram-negative bacterial pathogens. The structural classification of OAg, based on serological typing and sequence analysis, is important in epidemiology and the surveillance of outbreaks of bacterial infections. Despite the diverse chemical structures of OAg repeating units (RUs), the genetic basis of RU assembly remains poorly understood and represents a major limitation in assigning gene functions in polysaccharide biosynthesis. Here, we describe a genetic approach to interrogate the functional order of glycosyltransferases (GTs). Using Shigella flexneri as a model, we established an initial glycosyltransferase (IT)-controlled system, which allows functional order allocation of the subsequent GT in a 2-fold manner as follows: (i) first, by reporting the growth defects caused by the sequestration of UndP through disruption of late GTs and (ii) second, by comparing the molecular sizes of stalled OAg intermediates when each putative GT is disrupted. Using this approach, we demonstrate that for RfbF and RfbG, the GT involved in the assembly of S. flexneri backbone OAg RU, RfbG, is responsible for both the committed step of OAg synthesis and the third transferase for the second L-Rha. We also show that RfbF functions as the last GT to complete the S. flexneri OAg RU backbone. We propose that this simple and effective genetic approach can be also extended to define the functional order of enzymatic synthesis of other diverse polysaccharides produced both by Gram-negative and Gram-positive bacteria.IMPORTANCEThe genetic basis of enzymatic assembly of structurally diverse O antigen (OAg) repeating units (RUs) in Gram-negative pathogens is poorly understood, representing a major limitation in our understanding of gene functions for the synthesis of bacterial polysaccharides. We present a simple genetic approach to confidently assign glycosyltransferase (GT) functions and the order in which they act during assembly of the OAg RU. We employed this approach to determine the functional order of GTs involved in Shigella flexneri OAg assembly. This approach can be generally applied in interrogating GT functions encoded by other bacterial polysaccharides to advance our understanding of diverse gene functions in the biosynthesis of polysaccharides, key knowledge in advancing biosynthetic polysaccharide production.


Subject(s)
Bacterial Proteins , Glycosyltransferases , O Antigens , Shigella flexneri , Shigella flexneri/genetics , Shigella flexneri/enzymology , Shigella flexneri/metabolism , O Antigens/biosynthesis , O Antigens/genetics , O Antigens/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism
11.
Anal Bioanal Chem ; 416(16): 3687-3696, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38748247

ABSTRACT

Glycans participate in a vast number of recognition systems in diverse organisms in health and in disease. However, glycans cannot be sequenced because there is no sequencer technology that can fully characterize them. There is no "template" for replicating glycans as there are for amino acids and nucleic acids. Instead, glycans are synthesized by a complicated orchestration of multitudes of glycosyltransferases and glycosidases. Thus glycans can vary greatly in structure, but they are not genetically reproducible and are usually isolated in minute amounts. To characterize (sequence) the glycome (defined as the glycans in a particular organism, tissue, cell, or protein), glycosylation pathway prediction using in silico methods based on glycogene expression data, and glycosylation simulations have been attempted. Since many of the mammalian glycogenes have been identified and cloned, it has become possible to predict the glycan biosynthesis pathway in these systems. By then incorporating systems biology and bioprocessing technologies to these pathway models, given the right enzymatic parameters including enzyme and substrate concentrations and kinetic reaction parameters, it is possible to predict the potentially synthesized glycans in the pathway. This review presents information on the data resources that are currently available to enable in silico simulations of glycosylation and related pathways. Then some of the software tools that have been developed in the past to simulate and analyze glycosylation pathways will be described, followed by a summary and vision for the future developments and research directions in this area.


Subject(s)
Computer Simulation , Polysaccharides , Glycosylation , Polysaccharides/metabolism , Polysaccharides/chemistry , Animals , Humans , Software , Glycosyltransferases/metabolism
12.
Mol Microbiol ; 121(6): 1245-1261, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38750617

ABSTRACT

Linear, unbranched (1,3;1,4)-ß-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-ß-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-ß- and (1,4)-ß-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-ß-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.


Subject(s)
Glycosyltransferases , beta-Glucans , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , beta-Glucans/metabolism , Cell Wall/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Polysaccharides, Bacterial/biosynthesis , Polysaccharides, Bacterial/metabolism
13.
Carbohydr Polym ; 337: 122164, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38710558

ABSTRACT

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.


Subject(s)
Glucans , Glycosyltransferases , Water , Glucans/chemistry , Water/chemistry , Glycosyltransferases/metabolism , Glycosyltransferases/chemistry , Streptococcus/enzymology , Solubility , Streptococcus mutans/enzymology
14.
BMC Plant Biol ; 24(1): 400, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38745278

ABSTRACT

XTH genes are key genes that regulate the hydrolysis and recombination of XG components and plays role in the structure and composition of plant cell walls. Therefore, clarifying the changes that occur in XTHs during plant defense against abiotic stresses is informative for the study of the plant stress regulatory mechanism mediated by plant cell wall signals. XTH proteins in Arabidopsis thaliana was selected as the seed sequences in combination with its protein structural domains, 80 members of the BnXTH gene family were jointly identified from the whole genome of the Brassica napus ZS11, and analyzed for their encoded protein physicochemical properties, phylogenetic relationships, covariance relationships, and interoperating miRNAs. Based on the transcriptome data, the expression patterns of BnXTHs were analyzed in response to different abiotic stress treatments. The relative expression levels of some BnXTH genes under Al, alkali, salt, and drought treatments after 0, 6, 12 and 24 h were analyzed by using qRT-PCR to explore their roles in abiotic stress tolerance in B. napus. BnXTHs showed different expression patterns in response to different abiotic stress signals, indicating that the response mechanisms of oilseed rape against different abiotic stresses are also different. This paper provides a theoretical basis for clarifying the function and molecular genetic mechanism of the BnXTH gene family in abiotic stress tolerance in rapeseed.


Subject(s)
Brassica napus , Gene Expression Regulation, Plant , Glycosyltransferases , Multigene Family , Phylogeny , Stress, Physiological , Brassica napus/genetics , Brassica napus/enzymology , Stress, Physiological/genetics , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Genes, Plant , Arabidopsis/genetics , Arabidopsis/enzymology
15.
Nat Commun ; 15(1): 3792, 2024 May 06.
Article in English | MEDLINE | ID: mdl-38710711

ABSTRACT

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease.


Subject(s)
Protozoan Proteins , Toxoplasma , Toxoplasma/enzymology , Toxoplasma/genetics , Glycosylation , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/chemistry , Humans , Crystallography, X-Ray , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Cell Wall/metabolism , Animals
16.
Int J Biol Macromol ; 269(Pt 1): 131813, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38685537

ABSTRACT

Microbial exopolysaccharides (EPS) have various physiological functions such as antioxidant, anti-tumor, cholesterol lowering, and immune regulation. However, improving traditional fermentation conditions to increase the production of EPS from Lactiplantibacillus plantarum (L. plantarum) is limited. In this study, we aimed to better improve EPS production and physiological functions of L. plantarum YM-4-3 strain by overexpressing and knocking out the priming glycosyltransferase genes cps 2E and cps 4E for the first time. As a result, the EPS production of the overexpression strain was 30.15 %, 26.84 % and 36.29 % higher than WT, respectively. The EPS production of the knockout strain was significantly lower than that of the WT. At the same time, transcriptome data showed that the gene expression levels of each experimental strain had changed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways found that the glycolysis/gluconeogenesis pathway had the highest gene enrichment in the metabolic pathway. The monosaccharide components of the EPS of each experimental strain were different from those of the WT and the EPS of the experimental strain showed stronger activity against oxidation. In conclusion, this study contributes to the efficient production and application of L. plantarum EPS and helps to understand the mechanism of EPS regulation in L. plantarum.


Subject(s)
Glycosyltransferases , Lactobacillus plantarum , Polysaccharides, Bacterial , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Polysaccharides, Bacterial/biosynthesis , Lactobacillus plantarum/genetics , Lactobacillus plantarum/metabolism , Gene Expression Regulation, Bacterial , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Fermentation
17.
Angew Chem Int Ed Engl ; 63(25): e202402546, 2024 Jun 17.
Article in English | MEDLINE | ID: mdl-38616162

ABSTRACT

Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'-OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one-pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6-fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5-100 %, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhG biosynthesis, but also serves as a proof-of-concept for cell-free enzymatic construction of diverse natural products.


Subject(s)
Glycosides , Glycosyltransferases , Protein Engineering , Glycosides/chemistry , Glycosides/biosynthesis , Glycosides/metabolism , Glycosyltransferases/metabolism , Glycosyltransferases/chemistry , Catechol Oxidase/metabolism , Catechol Oxidase/chemistry
18.
Curr Opin Chem Biol ; 80: 102457, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38657391

ABSTRACT

Carbohydrate-active enzymes (CAZymes) are responsible for the biosynthesis, modification and degradation of all glycans in Nature. Advances in genomic and metagenomic methodologies, in conjunction with lower cost gene synthesis, have provided access to a steady stream of new CAZymes with both well-established and novel mechanisms. At the same time, increasing access to cryo-EM has resulted in exciting new structures, particularly of transmembrane glycosyltransferases of various sorts. This improved understanding has resulted in widespread progress in applications of CAZymes across diverse fields, including therapeutics, organ transplantation, foods, and biofuels. Herein, we highlight a few of the many important advances that have recently been made in the understanding and applications of CAZymes.


Subject(s)
Glycosyltransferases , Glycosyltransferases/metabolism , Humans , Animals , Enzymes/metabolism , Enzymes/chemistry , Polysaccharides/metabolism , Polysaccharides/chemistry , Carbohydrates/chemistry , Carbohydrate Metabolism
19.
J Microbiol Biotechnol ; 34(5): 1154-1163, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38563097

ABSTRACT

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-ß-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-ß-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-ß-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-ß-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.


Subject(s)
Apigenin , Corynebacterium glutamicum , Glucosides , Metabolic Engineering , Corynebacterium glutamicum/metabolism , Corynebacterium glutamicum/genetics , Apigenin/metabolism , Metabolic Engineering/methods , Glucosides/metabolism , Glucosides/biosynthesis , Glycosylation , Bacillus licheniformis/metabolism , Bacillus licheniformis/genetics , Bacillus licheniformis/enzymology , Uridine Diphosphate Glucose/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , UTP-Glucose-1-Phosphate Uridylyltransferase/metabolism , UTP-Glucose-1-Phosphate Uridylyltransferase/genetics , Glycosyltransferases/metabolism , Glycosyltransferases/genetics
20.
ACS Chem Biol ; 19(4): 992-998, 2024 04 19.
Article in English | MEDLINE | ID: mdl-38562012

ABSTRACT

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.


Subject(s)
Flagellin , Glycosyltransferases , Paenibacillus , Amino Acid Sequence , Cysteine/metabolism , Flagellin/metabolism , Glycosylation , Glycosyltransferases/metabolism , Paenibacillus/enzymology , Paenibacillus/metabolism
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