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1.
Physiol Plant ; 176(3): e14329, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38695156

RESUMO

Although tetraploid wheat has rich genetic variability for cultivar improvement, its physiological mechanisms associated with photosynthetic productivity and resilience under nitrogen (N) deficit stress have not been investigated. In this study, we selected emmer wheat (Kronos, tetraploid), Yangmai 25 (YM25, hexaploid), and Chinese Spring (CS, hexaploid) as materials and investigated the differences in net photosynthetic rate (Pn), carboxylation capacity, electron transfer capacity, photosynthetic product output, and photosynthetic N allocation under normal N (CK) and low N (LN) through hydroponic experiments. Tetraploid emmer wheat (Kronos) had a stronger photosynthetic capacity than hexaploid wheat (YM25, CS) under low N stress, which mainly associated with the higher degree of PSII opening, electron transfer rate, Rubisco content and activity, ATP/ADP ratio, Rubisco activase (Rca) activity and Rubisco activation state, and more leaves N allocation to the photosynthetic apparatus, especially the proportion of N allocation to carboxylation under low N stress. Moreover, Kronos reduced the feedback inhibition of photosynthesis by sucrose accumulation through higher sucrose phosphate synthetase (SPS) activity and triose phosphate utilization rate (VTPU). Overall, Kronos could allocate more N to the photosynthetic components to improve Rubisco content and activity to maintain photosynthetic capacity under low N stress while enhancing triose phosphate output to reduce feedback inhibition of photosynthesis. This study reveals the physiological mechanisms of emmer wheat that maintain the photosynthetic capacity under low N stress, which will provide indispensable germplasm resources for elite low-N-tolerant wheat improvement and breeding.


Assuntos
Nitrogênio , Fotossíntese , Ribulose-Bifosfato Carboxilase , Triticum , Fotossíntese/fisiologia , Triticum/fisiologia , Triticum/genética , Triticum/metabolismo , Nitrogênio/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Estresse Fisiológico , Folhas de Planta/fisiologia , Folhas de Planta/metabolismo , Adaptação Fisiológica , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Clorofila/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Glucosiltransferases/metabolismo , Glucosiltransferases/genética
2.
Biotechnol J ; 19(5): e2400178, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719574

RESUMO

Sucrose isomerase (SIase) catalyzes the hydrolysis and isomerization of sucrose into isomaltulose, a functional sugar extensively used in the food industry. However, the lack of safe and efficient heterologous expression systems for SIase has constrained its production and application. In this study, an engineered Bacillus subtilis strain for antibiotic-free SIase production was developed via a food-grade expression system. First, the B. subtilis strain TEA was modified through the CRISPR/Cas9 system, resulting in a mutant strain TEA4, which exhibited enhanced capabilities for recombinant protein expression. For efficient and safe production of SIase, different constitutive and inducible promoters were evaluated. The maltose-inducible promoter Poglv was found to have an extracellular SIase activity of 21.7 U mL-1 in engineered strain TEA4. Subsequent optimization of the culture medium further increased SIase activity to 26.4 U mL-1 during shake flask cultivation. Eventually, using the crude enzyme solution of the engineered strain in biotransformation reactions resulted in a high yield of isomaltulose under high concentrations sucrose, achieving a maximum yield of 83.1%. These findings demonstrated an engineered B. subtilis strain for antibiotic-free SIase production, paving the way for its scale-up industrial production and application.


Assuntos
Bacillus subtilis , Glucosiltransferases , Isomaltose , Proteínas Recombinantes , Sacarose , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Isomaltose/metabolismo , Isomaltose/análogos & derivados , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Sacarose/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Engenharia Metabólica/métodos , Regiões Promotoras Genéticas/genética , Sistemas CRISPR-Cas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
3.
Glycobiology ; 34(6)2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38690785

RESUMO

Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N-Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants.


Assuntos
Arabidopsis , Celulose , Glucosilceramidas , Glucosiltransferases , Arabidopsis/metabolismo , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Celulose/metabolismo , Celulose/biossíntese , Glucosilceramidas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , 1-Desoxinojirimicina/farmacologia , 1-Desoxinojirimicina/análogos & derivados , Parede Celular/metabolismo
4.
Plant Physiol Biochem ; 210: 108591, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38583314

RESUMO

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.


Assuntos
Cotilédone , Regulação da Expressão Gênica de Plantas , Glucosiltransferases , Lotus , Proteínas de Plantas , Sementes , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Cotilédone/genética , Cotilédone/metabolismo , Cotilédone/enzimologia , Lotus/genética , Lotus/enzimologia , Lotus/metabolismo , Sementes/genética , Sementes/metabolismo , Sementes/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Açúcares/metabolismo
5.
Appl Environ Microbiol ; 90(4): e0208723, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38557137

RESUMO

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.


Assuntos
Polissacarídeos , Streptomyces lividans , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Polissacarídeos/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Celulose/metabolismo
6.
Food Chem ; 448: 139182, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38569413

RESUMO

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1-16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The kcat/Km value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin.


Assuntos
Proteínas de Bactérias , Biotransformação , Deinococcus , Flavanonas , Glucosídeos , Glucosiltransferases , Inibidores de Glicosídeo Hidrolases , Flavanonas/metabolismo , Flavanonas/química , Deinococcus/enzimologia , Deinococcus/metabolismo , Deinococcus/química , Deinococcus/genética , Glucosiltransferases/metabolismo , Glucosiltransferases/química , Glucosiltransferases/genética , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glucosídeos/metabolismo , Glucosídeos/química , Simulação de Acoplamento Molecular , Cinética , alfa-Glucosidases/metabolismo , alfa-Glucosidases/química
7.
J Agric Food Chem ; 72(18): 10497-10505, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38659290

RESUMO

Despite their broad application potential, the widespread use of ß-1,3-glucans has been hampered by the high cost and heterogeneity associated with current production methods. To address this challenge, scalable and economically viable processes are needed for the production of ß-1,3-glucans with tailorable molecular mass distributions. Glycoside phosphorylases have shown to be promising catalysts for the bottom-up synthesis of ß-1,3-(oligo)glucans since they combine strict regioselectivity with a cheap donor substrate (i.e., α-glucose 1-phosphate). However, the need for an expensive priming substrate (e.g., laminaribiose) and the tendency to produce shorter oligosaccharides still form major bottlenecks. Here, we report the discovery and application of a thermostable ß-1,3-oligoglucan phosphorylase originating from Anaerolinea thermophila (AtßOGP). This enzyme combines a superior catalytic efficiency toward glucose as a priming substrate, high thermostability, and the ability to synthesize high molecular mass ß-1,3-glucans up to DP 75. Coupling of AtßOGP with a thermostable variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable ß-1,3-(oligo)glucans from sucrose, with a near-complete conversion of >99 mol %. This cost-efficient process for the conversion of renewable bulk sugar into ß-1,3-(oligo)glucans should facilitate the widespread application of these versatile functional fibers across various industries.


Assuntos
Proteínas de Bactérias , Estabilidade Enzimática , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , beta-Glucanas/química , beta-Glucanas/metabolismo , Bifidobacterium adolescentis/enzimologia , Bifidobacterium adolescentis/genética , Bifidobacterium adolescentis/química , Bifidobacterium adolescentis/metabolismo , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Especificidade por Substrato , Fosforilases/metabolismo , Fosforilases/química , Fosforilases/genética , Clostridiales/enzimologia , Clostridiales/genética , Clostridiales/química , Biocatálise , Temperatura Alta
8.
Bioorg Chem ; 146: 107287, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38503024

RESUMO

Enzyme-based glycosylation is of great interest in the context of natural products decoration. Yet, its industrial application is hindered by optimisation difficulties and hard-to-standardise productivities. In this study, five sugar nucleotide-dependent glucosyltransferases from different origins (bacterial, plant and fungal) were coupled with soy sucrose synthase (GmSuSy) to create a set of diverse cascade biocatalysts for flavonoid glucosylation, which evaluation brought new insights into the field. Investigations into co-expression conditions and reaction settings enabled to define optimal induction temperature (25 °C) and uridine diphosphate (UDP) concentration (0.5 mM) for all tested pairs of enzymes. Moreover, the influence of pH and substrate concentration on the monoglucosylated product distribution was detected and analysed. The utilisation of crude protein extracts as a cost-effective source of catalysts unveiled their glycosidase activity against flavonoid glucosides, resulting in decreased productivity, which, to our knowledge, has not previously been discussed in such a context. Additionally, examination of the commercially available EziG immobilisation resins showed that selection of suitable carrier for solid catalyst production can be problematic and not only enzyme's but also reagent's properties have to be considered. Flavonoids, due to their complexation and hydrophobic properties, can adsorb on different types of surfaces, including divalent metal ions required for IMAC based immobilisation, necessitating detailed examination of the resins while the catalysis design.


Assuntos
Flavonoides , Glucosiltransferases , Glucosiltransferases/metabolismo , Glicosilação , Nucleotídeos
9.
ACS Synth Biol ; 13(4): 1290-1302, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38526141

RESUMO

The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X-1-N-X+1-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X+2 position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.


Assuntos
Proteínas de Bactérias , Glicosiltransferases , Glicosilação , Glicosiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Glucosiltransferases/metabolismo , Peptídeos/metabolismo , Bactérias/metabolismo
10.
J Pathol ; 263(1): 8-21, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38332735

RESUMO

Pompe disease is a lysosomal storage disorder that preferentially affects muscles, and it is caused by GAA mutation coding acid alpha-glucosidase in lysosome and glycophagy deficiency. While the initial pathology of Pompe disease is glycogen accumulation in lysosomes, the special role of the lysosomal pathway in glycogen degradation is not fully understood. Hence, we investigated the characteristics of accumulated glycogen and the mechanism underlying glycophagy disturbance in Pompe disease. Skeletal muscle specimens were obtained from the affected sites of patients and mouse models with Pompe disease. Histological analysis, immunoblot analysis, immunofluorescence assay, and lysosome isolation were utilized to analyze the characteristics of accumulated glycogen. Cell culture, lentiviral infection, and the CRISPR/Cas9 approach were utilized to investigate the regulation of glycophagy accumulation. We demonstrated residual glycogen, which was distinguishable from mature glycogen by exposed glycogenin and more α-amylase resistance, accumulated in the skeletal muscle of Pompe disease. Lysosome isolation revealed glycogen-free glycogenin in wild type mouse lysosomes and variously sized glycogenin in Gaa-/- mouse lysosomes. Our study identified that a defect in the degradation of glycogenin-exposed residual glycogen in lysosomes was the fundamental pathological mechanism of Pompe disease. Meanwhile, glycogenin-exposed residual glycogen was absent in other glycogen storage diseases caused by cytoplasmic glycogenolysis deficiencies. In vitro, the generation of residual glycogen resulted from cytoplasmic glycogenolysis. Notably, the inhibition of glycogen phosphorylase led to a reduction in glycogenin-exposed residual glycogen and glycophagy accumulations in cellular models of Pompe disease. Therefore, the lysosomal hydrolysis pathway played a crucial role in the degradation of residual glycogen into glycogenin, which took place in tandem with cytoplasmic glycogenolysis. These findings may offer a novel substrate reduction therapeutic strategy for Pompe disease. © 2024 The Pathological Society of Great Britain and Ireland.


Assuntos
Doença de Depósito de Glicogênio Tipo II , Glicoproteínas , Humanos , Camundongos , Animais , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/patologia , Doença de Depósito de Glicogênio Tipo II/terapia , Glicogênio/análise , Glicogênio/metabolismo , Glucosiltransferases/metabolismo , Músculo Esquelético/patologia , Lisossomos/metabolismo
11.
World J Microbiol Biotechnol ; 40(4): 114, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38418710

RESUMO

Six lactic acid bacteria (LAB) isolated from Algerian sheep's milk, traditional butter, date palm sap and barley, which produce dextran, mannitol, oligosaccharides and vitamin B2 have been characterized. They were identified as Leuconostoc mesenteroides (A4X, Z36P, B12 and O9) and Liquorilactobacillus mali (BR201 and FR123). Their exopolysaccharides synthesized from sucrose by dextransucrase (Dsr) were characterized as dextrans with (1,6)-D-glucopyranose units in the main backbone and branched at positions O-4, O-2 and/or O-3, with D-glucopyranose units in the side chain. A4X was the best dextran producer (4.5 g/L), while the other strains synthesized 2.1-2.7 g/L. Zymograms revealed that L. mali strains have a single Dsr with a molecular weight (Mw) of ~ 145 kDa, while the Lc. mesenteroides possess one or two enzymes with 170-211 kDa Mw. As far as we know, this is the first detection of L. mali Dsr. Analysis of metabolic fluxes from sucrose revealed that the six LAB produced mannitol (~ 12 g/L). The co-addition of maltose-sucrose resulted in the production of panose (up to 37.53 mM), an oligosaccharide known for its prebiotic effect. A4X, Z36P and B12 showed dextranase hydrolytic enzymatic activity and were able to produce another trisaccharide, maltotriose, which is the first instance of a dextranase activity encoded by Lc. mesenteroides strains. Furthermore, B12 and O9 grew in the absence of riboflavin (vitamin B2) and synthesized this vitamin, in a defined medium at the level of ~ 220 µg/L. Therefore, these LAB, especially Lc. mesenteroides B12, are good candidates for the development of new fermented food biofortified with functional compounds.


Assuntos
Leuconostoc mesenteroides , Animais , Ovinos , Dextranos/metabolismo , Dextranase/química , Dextranase/metabolismo , Manitol/metabolismo , Mali , Glucosiltransferases/metabolismo , Oligossacarídeos/química , Sacarose/metabolismo , Vitaminas/metabolismo , Leuconostoc/metabolismo
12.
PLoS One ; 19(2): e0292149, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38358988

RESUMO

Plant cells possess robust and flexible cell walls composed primarily of cellulose, a polysaccharide that provides structural support and enables cell expansion. Cellulose is synthesised by the Cellulose Synthase A (CESA) catalytic subunits, which form cellulose synthase complexes (CSCs). While significant progress has been made in unravelling CSC function, the trafficking of CSCs and the involvement of post-translational modifications in cellulose synthesis remain poorly understood. In order to deepen our understanding of cellulose biosynthesis, this study utilised immunoprecipitation techniques with CESA6 as the bait protein to explore the CSC and its interactors. We have successfully identified the essential components of the CSC complex and, notably, uncovered novel interactors associated with CSC trafficking, post-translational modifications, and the coordination of cell wall synthesis. Moreover, we identified TIP GROWTH DEFECTIVE 1 (TIP1) protein S-acyl transferases (PATs) as an interactor of the CSC complex. We confirmed the interaction between TIP1 and the CSC complex through multiple independent approaches. Further analysis revealed that tip1 mutants exhibited stunted growth and reduced levels of crystalline cellulose in leaves. These findings suggest that TIP1 positively influences cellulose biosynthesis, potentially mediated by its role in the S-acylation of the CSC complex.


Assuntos
Aciltransferases , Proteínas de Arabidopsis , Arabidopsis , Celulose , Glucosiltransferases , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Glucosiltransferases/metabolismo , Aciltransferases/metabolismo
13.
Arch Biochem Biophys ; 753: 109926, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38346547

RESUMO

Of the more than 100 families of glycosyltransferases, family 1 glycosyltransferases catalyze glycosylation using uridine diphosphate (UDP)-sugar as a sugar donor and are thus referred to as UDP-sugar:glycosyl transferases. The blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin, flavone, and metal ions. Flavone 7-O-ß-glucoside-4'-O-ß-glucoside in the plant is sequentially biosynthesized from flavons by UDP-glucose:flavone 4'-O-glucosyltransferase (NmF4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (NmF4'G7GT). To identify the molecular mechanisms of glucosylation of flavone, the crystal structures of NmF4'G7GT in its apo form and in complex with UDP-glucose or luteolin were determined, and molecular structure prediction using AlphaFold2 was conducted for NmF4'GT. The crystal structures revealed that the size of the ligand-binding pocket and interaction environment for the glucose moiety at the pocket entrance plays a critical role in the substrate preference in NmF4'G7GT. The substrate specificity of NmF4'GT was examined by comparing its model structure with that of NmF4'G7GT. The structure of NmF4'GT may have a smaller acceptor pocket, leading to a substrate preference for non-glucosylated flavones (or flavone aglycones).


Assuntos
Flavonas , Glucosiltransferases , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Ligantes , Uridina Difosfato Glucose/química , Glucose , Glicosiltransferases , Glucosídeos , Especificidade por Substrato
14.
Traffic ; 25(1): e12927, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38272446

RESUMO

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.


Assuntos
Dobramento de Proteína , Doenças Raras , Animais , Humanos , Doenças Raras/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Retículo Endoplasmático/metabolismo , Mutação , Mamíferos/metabolismo , Glucosiltransferases/metabolismo
15.
Biotechnol Lett ; 46(2): 173-181, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38184486

RESUMO

OBJECTIVE: Salidroside is an important plant-derived aromatic compound with diverse biological properties. The main objective of this study was to synthesize salidroside from tyrosol using UDP-glucosyltransferase (UGT) with in situ regeneration of UDP-glucose (UDPG). RESULTS: The UDP-glucosyltransferase 85A1 (UGT85A1) from Arabidopsis thaliana, which showed high activity and regioselectivity towards tyrosol, was selected for the production of salidroside. Then, an in vitro cascade reaction for in situ regeneration of UDPG was constructed by coupling UGT85A1 to sucrose synthase from Glycine max (GmSuSy). The optimal UGT85A1-GmSuSy activity ratio of 1:2 was determined to balance the efficiency of salidroside production and UDP-glucose regeneration. Different cascade reaction conditions for salidroside production were also determined. Under the optimized condition, salidroside was produced at a titer of 6.0 g/L with a corresponding molar conversion of 99.6% and a specific productivity of 199.1 mg/L/h in a continuous feeding reactor. CONCLUSION: This is the highest salidroside titer ever reported so far using biocatalytic approach.


Assuntos
Glucosídeos , Glucosiltransferases , Fenóis , Álcool Feniletílico/análogos & derivados , Uridina Difosfato Glucose , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Biocatálise , Glucose
16.
Food Chem ; 442: 138432, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38241991

RESUMO

The fruit of Lycium barbarum (Lb), known as red goji berry, is a "superfruit" due to its abundance of bioactive compounds. Among these compounds, dicaffeoylspermidine derivatives (DCSPDs) have anti-oxidant and anti-Alzheimer's Disease activity. This study employed ultra-high-performance liquid chromatography with tandem mass spectrometry to investigate metabolic changes during the development and ripening stages of red goji berries. Totally 97 compounds, including 51 DCSPDs, were tentatively identified. Correlation analysis of these DCSPDs revealed that glycosyltransferases (GTs) play an important role in the formation of glycosylated DCSPDs. In vitro experiments characterized 3 novel GTs could add a glucosyl moiety to N1-caffeoyl-N10-dihydrocaffeoyl spermidine. Homologous GTs from L. ruthenicum (Lr) exhibited similar activity, despite the absence of abundant glycosylated DCSPDs in Lr. These findings provide insights into the metabolic changes and interconnections among active compounds in red goji berries. The identified GTs hold potential for metabolic engineering of DCSPDs and functional food development.


Assuntos
Lycium , Lycium/química , Frutas/química , Glucosiltransferases/metabolismo , Espectrometria de Massas em Tandem , Antioxidantes/química
17.
Int J Biol Macromol ; 259(Pt 2): 129315, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38211906

RESUMO

Cyclodextrin glycosyltransferase (CGTase) is a significant extracellular enzyme with diverse functions. CGTase is widely used in production of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch via transglycosylation reaction. Recent discoveries of novel CGTases from different microorganisms have expanded its applications but natural CGTase have lower yield, leading to heterologous expression for increased production to meet various needs. Moreover, significant advancements in directed evolution approach have been explored to alter the molecular structure of CGTase to enhance its performance. This review comprehensively summarizes the strategies employed in heterologous expression to boost CGTase production and secretion in various host. It also outlines molecular engineering approaches aimed to improving CGTase properties, including product and substrate specificity, catalytic efficiency, and thermal stability. Additionally, a considerable stability against changes in temperature and organic solvents can be obtained by immobilization.


Assuntos
Ciclodextrinas , Ciclodextrinas/química , Glucosiltransferases/metabolismo , Amido/metabolismo , Temperatura
18.
J Biosci Bioeng ; 137(1): 47-53, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38036317

RESUMO

Our pursuit of new compounds with enhanced bioavailability and bioactivity prompted us to employ the biotransformation-guided purification (BGP) approach which leverages proficient in vitro biotransformation techniques. Angelica dahurica roots, also called Baizhi in Chinese traditional medicine, are famous for their anti-inflammatory and analgesic properties. Herein, we applied the BGP methodology to Baizhi extracts, employing Deinococcus geothermalis amylosucrase (DgAS), an enzyme demonstrating catalytic competence across diverse substrates, for biotransformation. Initiating with a 70 % methanol extraction, we obtained the crude extract of commercial Baizhi powder, followed by an additional extraction using ethyl acetate. Notably, reactions performed on this extract yielded limited quantities of novel compounds. Subsequently, the extract underwent partitioning into four fractions based on HPLC profiling, leading to the successful isolation of a compound with significant yield from fraction 2 mixtures upon reaction with DgAS. Structural elucidation confirmed the compound as byakangelicin-7″-O-α-glucopyranoside (BG-G), a new alpha glycoside derivative of byakangelicin. Furthermore, validation experiments verified the capacity of DgAS to glycosylate pure byakangelicin, yielding BG-G. Remarkably, the aqueous solubility of BG-G exceeded that of byakangelicin by over 29,000-fold. In conclusion, BGP emerges as a potent strategy combining traditional medicinal insights with robust enzymatic tools for generating new compounds.


Assuntos
Glicosídeos , Medicina Tradicional Chinesa , Glucosiltransferases/metabolismo , Biotransformação
19.
Biotechnol Bioeng ; 121(2): 580-592, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37983971

RESUMO

One-pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α-d-glucose 1-phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step-by-step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial-rate models of the two-substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two-site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks.


Assuntos
Celobiose , Glucosiltransferases , Glucosiltransferases/metabolismo , Fosforilases/metabolismo , Glucose , Dissacarídeos , Sacarose , Cinética , Fosfatos , Especificidade por Substrato
20.
Nat Rev Mol Cell Biol ; 25(5): 340-358, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38102449

RESUMO

Plant cells build nanofibrillar walls that are central to plant growth, morphogenesis and mechanics. Starting from simple sugars, three groups of polysaccharides, namely, cellulose, hemicelluloses and pectins, with very different physical properties are assembled by the cell to make a strong yet extensible wall. This Review describes the physics of wall growth and its regulation by cellular processes such as cellulose production by cellulose synthase, modulation of wall pH by plasma membrane H+-ATPase, wall loosening by expansin and signalling by plant hormones such as auxin and brassinosteroid. In addition, this Review discusses the nuanced roles, properties and interactions of cellulose, matrix polysaccharides and cell wall proteins and describes how wall stress and wall loosening cooperatively result in cell wall growth.


Assuntos
Parede Celular , Celulose , Células Vegetais , Parede Celular/metabolismo , Celulose/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Desenvolvimento Vegetal/fisiologia , Plantas/metabolismo , Polissacarídeos/metabolismo , Glucosiltransferases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais
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