RESUMEN
Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.
Asunto(s)
Populus , Populus/genética , Populus/metabolismo , Xilanos/metabolismo , Acetilación , Biomasa , Biocombustibles/análisis , Plantas/metabolismo , Pared Celular/metabolismo , Lignina/metabolismoRESUMEN
Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.
Asunto(s)
Proteínas Bacterianas , Paenibacillus , Saccharum , Xilanos , Xilosa , Xilosidasas , Xilanos/metabolismo , Paenibacillus/metabolismo , Paenibacillus/enzimología , Proteínas Bacterianas/metabolismo , Saccharum/metabolismo , Saccharum/química , Xilosidasas/metabolismo , Xilosa/metabolismo , Reactores Biológicos/microbiología , Fibras de la Dieta/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Disacáridos/metabolismo , Glicósido Hidrolasas/metabolismoRESUMEN
Glycosyl composition and linkage analyses are important first steps toward understanding the structural diversity and biological importance of polysaccharides. Failure to fully solubilize samples prior to analysis results in the generation of incomplete and poor-quality composition and linkage data by gas chromatography-mass spectrometry (GC-MS). Acidic polysaccharides also do not give accurate linkage results, because they are poorly soluble in DMSO and tend to undergo ß-elimination during permethylation. Ionic liquids can solubilize polysaccharides, improving their derivatization and extraction for analysis. We show that water-insoluble polysaccharides become much more amenable to chemical analysis by first acetylating them in an ionic liquid. Once acetylated, these polysaccharides, having been deprived of their intermolecular hydrogen bonds, are hydrolyzed more readily for glycosyl composition analysis or methylated more efficiently for glycosyl linkage analysis. Acetylation in an ionic liquid greatly improves composition analysis of insoluble polysaccharides when compared to analysis without acetylation, enabling complete composition determination of normally recalcitrant polysaccharides. We also present a protocol for uronic acid linkage analysis that incorporates this preacetylation step. This protocol produces partially methylated alditol acetate derivatives in high yield with minimal ß-elimination and gives sensitive linkage results for acidic polysaccharides that more accurately reflect the structures being analyzed. We use important plant polysaccharides to show that the preacetylation step leads to superior results compared to traditional methodologies.
Asunto(s)
Líquidos Iónicos , Acetilación , Cromatografía de Gases y Espectrometría de Masas , Procesamiento Proteico-Postraduccional , PolisacáridosRESUMEN
Switchgrass, a forage and bioenergy crop, occurs as two main ecotypes with different but overlapping ranges of adaptation. The two ecotypes differ in a range of characteristics, including flowering time. Flowering time determines the duration of vegetative development and therefore biomass accumulation, a key trait in bioenergy crops. No causal variants for flowering time differences between switchgrass ecotypes have, as yet, been identified. In this study, we mapped a robust flowering time quantitative trait locus (QTL) on chromosome 4K in a biparental F2 population and characterized the flowering-associated transcription factor gene PvHd1, an ortholog of CONSTANS in Arabidopsis and Heading date 1 in rice, as the underlying causal gene. Protein modeling predicted that a serine to glycine substitution at position 35 (p.S35G) in B-Box domain 1 greatly altered the global structure of the PvHd1 protein. The predicted variation in protein compactness was supported in vitro by a 4 °C shift in denaturation temperature. Overexpressing the PvHd1-p.35S allele in a late-flowering CONSTANS-null Arabidopsis mutant rescued earlier flowering, whereas PvHd1-p.35G had a reduced ability to promote flowering, demonstrating that the structural variation led to functional divergence. Our findings provide us with a tool to manipulate the timing of floral transition in switchgrass cultivars and, potentially, expand their cultivation range.
Asunto(s)
Arabidopsis , Panicum , Panicum/genética , Arabidopsis/genética , Sitios de Carácter Cuantitativo , Fenotipo , Aminoácidos/genética , Flores/genéticaRESUMEN
In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the ß-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual ß-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent ß-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/enzimología , Pared Celular/metabolismo , Glucanos/metabolismo , Glucosiltransferasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Biocatálisis/efectos de los fármacos , Pared Celular/efectos de los fármacos , Detergentes/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glucosiltransferasas/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocótilo/efectos de los fármacos , Hipocótilo/crecimiento & desarrollo , Mutación/genética , Regiones Promotoras Genéticas/genética , Dominios Proteicos , Proteolípidos/metabolismo , SolubilidadRESUMEN
Xylans are a major component of plant cell walls. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis (Arabidopsis thaliana) xylan O-acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2-O-acetylation of xylan, followed by nonenzymatic acetyl migration to the O-3 position, resulting in products that are monoacetylated at both O-2 and O-3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/ß/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac-Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Polisacáridos/metabolismo , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arginina/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana , Modelos Moleculares , Mutación , Conformación Proteica , Xilanos/metabolismoRESUMEN
Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo-enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase-mediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl-transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.
Asunto(s)
Arabidopsis , Xilanos , Xilanos/química , Espectrometría de Masas en Tándem , Xilosa , Oligosacáridos/química , GlucuronosiltransferasaRESUMEN
This corrects the article DOI: 10.1038/nature21725.
RESUMEN
Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose-binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Populus/genética , Saccharomycetales/patogenicidad , Transcriptoma , Alelos , Mapeo Cromosómico , Cromosomas de las Plantas/química , Resistencia a la Enfermedad/genética , Perfilación de la Expresión Génica , Sitios Genéticos , Interacciones Huésped-Patógeno/inmunología , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/inmunología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Proteínas de Plantas/inmunología , Populus/inmunología , Populus/microbiología , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Saccharomycetales/fisiologíaRESUMEN
Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized "on chip" by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.
Asunto(s)
Glicosiltransferasas/química , Plantas/enzimología , Polisacáridos/análisis , Pared Celular/químicaRESUMEN
Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration-dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Glucuronosiltransferasa/metabolismo , Pectinas/biosíntesis , Arabidopsis/enzimología , Proteínas de Arabidopsis/química , Glucuronosiltransferasa/química , Células HEK293 , Humanos , Modelos Biológicos , Estructura Molecular , Pectinas/química , Electricidad Estática , Especificidad por Sustrato , Azúcares de Uridina Difosfato/metabolismoRESUMEN
The mechanistic underpinnings of the complex process of plant polysaccharide biosynthesis are poorly understood, largely because of the resistance of glycosyltransferase (GT) enzymes to structural characterization. In Arabidopsis thaliana, a glycosyl transferase family 37 (GT37) fucosyltransferase 1 (AtFUT1) catalyzes the regiospecific transfer of terminal 1,2-fucosyl residues to xyloglucan side chains - a key step in the biosynthesis of fucosylated sidechains of galactoxyloglucan. We unravel the mechanistic basis for fucosylation by AtFUT1 with a multipronged approach involving protein expression, X-ray crystallography, mutagenesis experiments and molecular simulations. Mammalian cell culture expressions enable the sufficient production of the enzyme for X-ray crystallography, which reveals the structural architecture of AtFUT1 in complex with bound donor and acceptor substrate analogs. The lack of an appropriately positioned active site residue as a catalytic base leads us to propose an atypical water-mediated fucosylation mechanism facilitated by an H-bonded network, which is corroborated by mutagenesis experiments as well as detailed atomistic simulations.
Asunto(s)
Arabidopsis/enzimología , Fucosiltransferasas/química , Glucanos/química , Modelos Estructurales , Xilanos/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografía por Rayos X , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Glicosilación , Simulación de Dinámica Molecular , Mutagénesis , Agua/metabolismo , Galactósido 2-alfa-L-FucosiltransferasaRESUMEN
The plant cell wall is a cellular exoskeleton consisting predominantly of a complex polysaccharide network that defines the shape of cells. During growth, this network can be loosened through the action of xyloglucan endotransglycosylases (XETs), glycoside hydrolases that "cut and paste" xyloglucan polysaccharides through a transglycosylation process. We have analyzed cohorts of XETs in different plant species to evaluate the substrate specificities of xyloglucan acceptors by using a set of synthetic oligosaccharides obtained by automated glycan assembly. The ability of XETs to incorporate the oligosaccharides into polysaccharides printed as microarrays and into stem sections of Arabidopsis thaliana, beans, and peas was assessed. We found that single xylose substitutions are sufficient for transfer, and xylosylation of the terminal glucose residue is not required by XETs, independent of plant species. To obtain information on the potential xylosylation pattern of the natural acceptor of XETs, that is, the nonreducing end of xyloglucan, we further tested the activity of xyloglucan xylosyl transferase (XXT) 2 on the synthetic xyloglucan oligosaccharides. These data shed light on inconsistencies between previous studies towards determining the acceptor substrate specificities of XETs and have important implications for further understanding plant cell wall polysaccharide synthesis and remodeling.
Asunto(s)
Pared Celular/metabolismo , Glicosiltransferasas/metabolismo , Oligosacáridos/metabolismo , Cromatografía Líquida de Alta Presión , Plantas/clasificación , Plantas/metabolismo , Especificidad de la EspecieRESUMEN
We report the automated glycan assembly of oligosaccharides related to the plant cell wall hemicellulosic polysaccharide xyloglucan. The synthesis of galactosylated xyloglucan oligosaccharides was enabled by introducing p-methoxybenzyl (PMB) as a temporary protecting group for automated glycan assembly. The generated oligosaccharides were printed as microarrays, and the binding of a collection of xyloglucan-directed monoclonal antibodies (mAbs) to the oligosaccharides was assessed. We also demonstrated that the printed glycans can be further enzymatically modified while appended to the microarray surface by Arabidopsis thaliana xyloglucan xylosyltransferase 2 (AtXXT2).
Asunto(s)
Anticuerpos Monoclonales/química , Arabidopsis/química , Automatización , Pared Celular/química , Oligosacáridos/síntesis química , Polisacáridos/química , Arabidopsis/enzimología , Pared Celular/enzimología , Análisis por Micromatrices , Oligosacáridos/química , Oligosacáridos/metabolismo , Pentosiltransferasa/metabolismo , Polisacáridos/metabolismoRESUMEN
Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far-reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10-L (IRX10-L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase, we have resolved two long-standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Xilanos/biosíntesis , Acetilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Celulosa/química , Glucosiltransferasas/química , Glucosiltransferasas/genética , Microfibrillas/química , Mutación/genética , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Proteínas de Arabidopsis/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Celulosa/biosíntesis , Cristalización , Resistencia a Medicamentos/efectos de los fármacos , Genes Dominantes/genética , Glucosiltransferasas/metabolismo , Espectroscopía de Resonancia Magnética , Microfibrillas/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Transporte de Proteínas/efectos de los fármacos , Quinolinas/química , Quinolinas/farmacología , Relación Estructura-ActividadRESUMEN
The hemicellulose 4-O-methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4-O-methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl-L-methionine to O-4 of α-D-glucopyranosyluronic acid residues that are linked to O-2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co(2+) for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4-O-methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O-methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O-methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Ácido Glucurónico/metabolismo , Metiltransferasas/metabolismo , Xilanos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Catálisis , Cationes/metabolismo , Pared Celular/enzimología , Éteres/metabolismo , Aparato de Golgi/metabolismo , Lignina/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Mutagénesis/fisiología , Polisacáridos/metabolismo , Estructura Terciaria de Proteína/fisiología , Xilanos/biosíntesisRESUMEN
O-Acetyl esterification is an important structural and functional feature of pectins present in the cell walls of all land plants. The amount and positions of pectin acetyl substituents varies across plant tissues and stages of development. Plant growth and response to biotic and abiotic stress are known to be significantly influenced by pectin O-acetylation. Gel formation is a key characteristic of pectins, and many studies have shown that gel formation is dependent upon the degree of acetylation. Previous studies have indicated that members of the TRICHOME BIREFRINGENCE-LIKE (TBL) family may play a role in the O-acetylation of pectin, however, biochemical evidence for acceptor specific pectin acetyltransferase activity remains to be confirmed and the exact mechanism(s) for catalysis must be determined. Pectin acetylesterases (PAEs) affect pectin acetylation as they hydrolyze acetylester bonds and have a role in the amount and distribution of O-acetylation. Several mutant studies suggest the critical role of pectin O-acetylation; however, additional research is required to fully understand this. This review aims to discuss the importance, role, and putative mechanism of pectin O-acetylation.
RESUMEN
â¢Xylan is an abundant carbohydrate component of plant cell walls that is vital for proper cell wall structure and vascular tissue development.â¢Xylan structure is known to vary between different tissues and species.â¢The role of xylan in the plant cell wall is to interact with cellulose, lignin, and hemicelluloses.â¢Xylan synthesis is directed by several types of Golgi-localized enzymes.â¢Xylan is being explored as an eco-friendly resource for diverse commercial applications.
RESUMEN
Glycosyltransferases (GTs) are carbohydrate-active enzymes that are encoded by the genomes of organisms spanning all domains of life. GTs catalyze glycosidic bond formation, transferring a sugar monomer from an activated donor to an acceptor substrate, often another saccharide. GTs from family 47 (GT47, PF03016) are involved in the synthesis of complex glycoproteins in mammals and insects and play a major role in the synthesis of almost every class of polysaccharide in plants, with the exception of cellulose, callose, and mixed linkage ß-1,3/1,4-glucan. GT47 enzymes adopt a GT-B fold and catalyze the formation of glycosidic bonds through an inverting mechanism. Unlike animal genomes, which encode few GT47 enzymes, plant genomes contain 30 or more diverse GT47 coding sequences. Our current knowledge of the GT47 family across plant species brings us an interesting view, showcasing how members exhibit a great diversity in both donor and acceptor substrate specificity, even for members that are classified in the same phylogenetic clade. Thus, we discuss how plant GT47 family members represent a great case to study the relationship between substrate specificity, protein structure, and protein evolution. Most of the plant GT47 enzymes that are identified to date are involved in biosynthesis of plant cell wall polysaccharides, including xyloglucan, xylan, mannan, and pectins. This indicates unique and crucial roles of plant GT47 enzymes in cell wall formation. The aim of this review is to summarize findings about GT47 enzymes and highlight new challenges and approaches on the horizon to study this family.