Arabinose Side Chains Research Articles

Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP’s richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Ultrasound-assisted enzymatic extraction, structural characterization, and anticancer activity of polysaccharides from Rosa roxburghii Tratt fruit

In this work, Rosa roxburghii Tratt fruit polysaccharides (RPs) were extracted by ultrasound-assisted enzymatic method. The highest extraction yield of RPs was 4.78 ± 0.10 % under the optimal extraction conditions. Two purified fractions named RP1 and RP3 were obtained and systematically characterized by a combination strategy of FT-IR, monosaccharide composition, molecular weight distribution, methylation and 2D NMR spectroscopy analyses. Structural analysis showed that the main chain of RP1 was composed of rhamnogalacturonan type I (RG-I), while the side chains were rich in arabinogalactan and galactose. RP3 was composed of long homogalacturonan (HG) backbone interspersed with alternating sequences of RG-I domains, with galactose and arabinose side chains. RP1 and RP3 induced apoptosis of MCF-7 cells in a dose dependent manner in vitro especially for RP1, and had no effect on L929 cells. Furthermore, the possible anticancer mechanisms were revealed, and results suggested that RP1 induced apoptosis through ROS-dependent pathway and mitochondrial pathway. The results of this work not only provided an efficient extraction method and theoretical basis for the application of RPs, but also may contribute to develop novel functional foods or pharmaceutical products for the prevention and treatment of human breast cancer disease.

Structural characterization of pectic polysaccharides from Amaranth caudatus leaves and the promotion effect on hippocampal glucagon-like peptide-1 level

In this study, decolorized pectic polysaccharides (D-ACLP) with molecular weight (Mw) distribution of 3483– 2,023,656 Da were prepared from Amaranth caudatus leaves. Purified polysaccharides (P-ACLP) with the Mw of 152,955 Da were further isolated from D-ACLP through gel filtration. The structure of P-ACLP was analyzed by 1D and 2D NMR spectra. P-ACLP were identified as rhamnogalacturonan-I (RG-I) containing dimeric arabinose side chains. The main chain of P-ACLP was composed of →4)-α-GalpA-(1→, →2)-β-Rhap-(1→, →3)-β-Galp-(1→ and →6)-β-Galp-(1→. There was a branched chain of α-Araf-(1→2)-α-Araf-(1→ connected to the O-6 position of →3)-β-Galp-(1→. The GalpA residues were partially methyl esterified at O-6 and acetylated at O-3. The 28-day consecutive gavage of D-ALCP (400 mg/kg) significantly elevated the hippocampal glucagon-like peptide-1 (GLP-1) levels in rats. The concentrations of butyric acid and total short chain fatty acids in the cecum contents also increased significantly. Moreover, D-ACLP could significantly increase the gut microbiota diversity and dramatically up-regulated the abundance of Actinobacteriota (phylum) and unclassified Oscillospiraceae (genus) in intestinal bacteria. Taking together, D-ACLP might promote the hippocampal GLP-1 level through the beneficial regulation of butyric acid-producing bacteria in gut microbiota. This study contributed to making full use of Amaranth caudatus leaves for cognitive dysfunction intervention in food industry.

Tailoring structural, rheological and gelling properties of watermelon rind pectin by enzymatic treatments

In this work, pectin extracts from watermelon rind (WRP) were enzymatically treated to evaluate their potential for preparing hydrogels with the addition of CaCl 2 . Based on a previous work, two different conditions were selected to obtain WRP extracts according to the 1) highest yield (OP) or 2) highest yield without negatively affecting the branching and native structure of pectin (OPA). Firstly, both WRP extracts were enzymatically modified using different treatments (de-esterification and/or de-branching of galacturonic and arabinose side chains, and deproteinization), and their impact on the esterification degree, monosaccharide composition and changes on their structural properties (linearity and branching degree) were deeply analysed. Then, the effect of the structural properties of the resulting pectin on the rheological behaviour and nanostructure of the hydrogels was investigated. The presence of long branched side chains and high methyl-esterified galacturonic acid chains promoted the formation of weaker hydrogels whereas de-esterification of the original pectin enabled intermolecular association giving rise to stronger hydrogels with the formation of ordered and densely packed structures (as deduced from SAXS results). However, the presence of small arabinogalactans side chains in the de-branched and de-esterified pectin extracts acted as reinforcement agents, inducing the formation of more densely packed networks and stronger hydrogels than their less-branched counterpart. These results demonstrated the impact of the pectin structure on the hydrogel-forming capacity. • Two watermelon rind pectin extracts were enzymatically modified. • Enzymes acting on neutral sugar side chains did not significantly de-branch the pectin structure. • Weaker gels were obtained when pectins were treated with de-branching enzymes. • Stronger gels were produced when pectin was de-esterified. • The strongest gels were produced when de-esterification and de-branching enzymes were combined.

Overcoming challenges in the synthesis of a lignin-carbohydrate complex (LCC) model: Mitsunobu versus Appel product

Arabinoxylan ferulate representing a macromolecular LCC model valid for annual plants is synthesized under Mitsunobu conditions. The content of ferulic acid ester is tuned by the reaction conditions achieving degree of substitution values from 0.09 to 0.45. Utilization of the chloride-free solvent N-methyl-2-pyrrolidone allows the design of pure Mitsunobu products without occurrence of deoxychloro moieties arising from Appel type reaction. 2D NMR experiments reveal nature-identical structure of ferulate moieties present at position 5 of the arabinose side chain. Enzymatic dehydrogenation polymerization of coniferyl alcohol on ferulate anchor groups under homogeneous conditions lead to beta -O-4, beta -5, and Hibbert ketone structures identified by Py-GC-MS. The results are valuable to study structure-property relationships within the formation of natural and non-native lignins.Graphical abstract

Arabinan hydrolysis by GH43 enzymes of Hungateiclostridium clariflavum and the potential synergistic mechanisms.

Glycoside hydrolase family 43 (GH43) represents a major source of arabinan- and arabinoxylan-active enzymes. Interestingly, some microbes remarkably enriched GH genes of this family, with the reason unknown. Hungateiclostridium clariflavum DSM 19,732 is an efficient lignocellulose degrader, which harbors up to 7 GH43 genes in its genome. We cloned three of the seven GH43 genes, and found that Abn43A is a unique endoarabinanase, which unprecedently showed approximately two times larger activity on sugar beet arabinan (116.8 U/mg) than that on linear arabinan, and it is efficient in arabinooligosaccharide production. Abn43B is an exoarabinanase which directly releases arabinose from linear arabinan. Abn43C is an α-L-arabinofuranosidase which is capable of splitting the arabinose side-chains from arabinooligosaccharides, arabinoxylooligosaccharides, and arabinoxylan. Most importantly, the three GH43 enzymes synergized in hydrolyzing arabinan. Compared to Abn43B alone, a supplement of Abn43A increased the arabinose production from linear arabinan by 150%, reaching 0.44g/g arabinan. Moreover, an addition of Abn43C to Abn43A and Abn43B boosted the arabinose production from sugar beet arabinan by 15 times, reaching 0.262g/g arabinan. Our work suggested the intensified functions of multiple GH43 enzymes toward arabinan degradation in H. clariflavum, and a potential synergetic mechanism among the three GH43 enzymes is suggested. KEY POINTS: • Endoarabinanase GH43A prefers branched substrate to linear one • Exoarabinanase GH43B can directly release arabinose from linear arabinan • The three GH43 enzymes synergized in arabinan hydrolysis.

High-efficiency separation of hemicellulose from bamboo by one-step freeze–thaw-assisted alkali treatment

The selectivity of alkali treatment (AT) for hemicellulose separation is reduced due to the alkali solubility of lignin. It was improved using freeze–thaw-assisted alkaline treatment (FT/AT). In this study, bamboo hemicellulose was separated via a one-step freeze–thaw-assisted alkali treatment (OFT/AT). The effects of freezing temperature, freezing time, alkali concentration, and treatment time on bamboo components were studied. The separation yield of hemicellulose was 73.26%, compared to 64.00% using conventional FT/AT. The separation of lignin and cellulose was inhibited as alkali concentration decreased from 7.0% to 5.0%. The extraction yield of hemicellulose increased from 46.35% to 56.12%. Structural analysis of extracted hemicellulose revealed the effective inhibition of the breakage of the xylose backbone and arabinose side chain of hemicellulose. This indicated that the molecular structure of extracted hemicellulose was relatively complete. It provides theoretical support for the efficient separation of hemicellulose by AT.

Experimental and theoretical investigation on interactions between xylose-containing hemicelluloses and procyanidins

During processing of plant-based foods, cell wall polysaccharides and polyphenols, such as procyanidins, interact extensively, thereby affecting their physicochemical properties along with their potential health effects. Although hemicelluloses are second only to pectins in affinity for procyanidins in cell walls, a detailed study of their interactions lacks. We investigated the interactions between representative xylose-containing water-soluble hemicelluloses and procyanidins. Turbidity, ITC and DLS were used to determine the relative affinities, and theoretical calculations further ascertained the interactions mechanisms. Xyloglucan and xylan exhibited respectively the strongest and weakest interactions with procyanidins. The different arabinoxylans interacted with procyanidins in a similar strength, intermediate between xyloglucans and xylans. Therefore, the strength of the interaction depended on the structure itself rather than on some incidental properties, e.g., viscosity and molar mass. The arabinose side-chain of arabinoxylan did not inhibit interactions. The computational investigation corroborated the experimental results in that the region of interaction between xyloglucan and procyanidins was significantly wider than that of other hemicelluloses.

Structure and fermentation characteristics of five polysaccharides sequentially extracted from sugar beet pulp by different methods

Pectic polysaccharides can serve as the best alternative for prebiotics, as they can be fermented in the gut by the microbiota. Many reports suggest that pectic polysaccharides' fermentation characteristics are superior to commercially available prebiotics as Inulin. However, these researches have focused on commercial pectin commonly extracted from apples or citrus, while pectins can exhibit complex and diverse structures. In this study, we extracted and characterized five structurally different polysaccharides from sugar beet pulp by water, 1,2-Cyclohexanediaminetetraacetic acid (CDTA), sodium carbonate, and sodium hydroxide, and then assessed their impacts on the human fecal microbiome. CDTA-soluble fraction and sodium carbonate-soluble fraction were identified as pectic polysaccharides, while water-soluble fraction and sodium hydroxide-soluble fractions were rich in the arabinose side chain. All polysaccharides were fermented more easily than inulin. Some neutral sugars were more easily fermented than acid sugar, and arabinose may be the first monosaccharide to be utilized by the microbiota, followed by glucose, fucose and galacturonic acid. After 48h of fermentation, some health-promoting bacteria like Faecalibacterium, Bifidobacterium, Lactobacillus, Blautia, and Prevotella increased, while some pathogenic bacteria like Fusobacterium, Sutterella, and Dorea decreased in all polysaccharides and Inulin. Hereby, through the study of the correlation between monosaccharide composition and microbiota, we showed that monosaccharide composition might be a critical structural feature linked to the polysaccharide-microbiota modulating effect. This could be helpful to have a criterion to choose the polysaccharides to better modulate the microbiota.

Influence of pH and ionic strength on the aggregation behaviors of xylan rich hemicelluloses with alkaline lignins

The evolution of xylan-rich hemicelluloses (XH) aggregation behaviors in the presence of alkaline lignins (AL) under a wide range of pH values and NaCl concentration were investigated via dynamic light scattering and turbidity measurements. XH isolated from wheat straw contain a xylose backbone with arabinose side chains and a small amount of phenol groups. XH tend to aggregate in solution due to their low ratio of arabinose to xylose and hydrophobic phenol groups. AL interact with XH through the phenol groups bonded to the hemicellulose main chain to form an AL-XH complex. As the pH value decreases, the particle size and turbidity of AL, XH and their bonded complex all increase. The size of the AL-XH complex agglomerate is greater than the size of a XH at the same pH value, which indicates that the self-assembly of lignin molecules initiate the aggregation of XH. The particle size and turbidity of XH and AL-XH complexes increase as the XH concentration increase. At low pH values, e.g., 6.0, the particle size of the AL-XH complex more obviously increases compared to the XH particles. The size and turbidity of the AL, XH, and AL-XH complex agglomerates increased as the NaCl concentration increased.

Transgenic rice plants expressing the α-L-arabinofuranosidase of Coprinopsis cinerea exhibit strong dwarfism and markedly enhanced tillering.

Lignocellulosic materials are potential renewable sources of fermentable sugars for bioethanol production. In this study, we used the CcAbf62A gene encoding CcAbf62A, a putative extracellular α-L-arabinofuranosidase, cloned from the mycotrophic basidiomycete Coprinopsis cinerea. CcAbf62A acts on arabinoxylan, the major hemicellulose of grasses, releasing arabinose. CcAbf62A was introduced into rice with the aim of enhancing delignification efficiency and the availability of lignocellulosic materials without reducing lignin content. Among the 32 lines of regenerated transgenic rice, 13 exhibited markedly disrupted elongation growth and excessive tillering (dwarf), seven showed delayed elongation growth (retarded-growth), and 12 showed phenotypes similar to those of control plants (normal). Additionally, the dwarf lines showed reduced acclimation. RT-PCR analysis revealed that dwarf lines had higher levels of CcAbf62A expression than retarded-growth and normal lines. Although the lignin content of transgenic rice plants expressing CcAbf62A did not differ significantly from that of control rice plants, dwarf lines were characterized by delayed deposition of lignin in the culms compared with the controls. The reduced acclimation ability of dwarf lines is believed to be associated with increased water loss and reduced water conductivity concomitant with delayed lignin deposition. Contrary to expectations, the alkaline delignification rates of dwarf and retarded-growth Abf lines were slightly lower than those of control rice plants. Our findings indicate that CcAbf62A reduces ferulate-lignin cross-links by detaching arabinose side chains from arabinoxylan and increases the relative abundance of alkaline-resistant benzyl ether cross-links. CcAbf62A is anticipated to provide new approaches for breeding plants containing altered lignocellulosic materials or lodging-resistant crops.

Extraction and characterization of lipids and phenolic compounds from the brans of different wheat varieties

The lipids and phenolic compounds of wheat bran (WB) and hemicellulose B (water extractable Hemi. B and alkali extractable Hemi. B) fractions from four wheat varieties were investigated. The X-ray diffraction pattern and NMR spectra of those Hemi. B were also studied. The predominant lipid components identified in WB include triacylglycerols (41.58–53.24 wt %) and free fatty acids (7.62–13.85 wt %) with the highest content in HD-2967 and C-306 varieties (9.13 and 13.85 wt %, respectively). The WE-Hemi. B had more lipids than the AE-Hemi. B fractions. The HD-3086 variety had the highest lipid content in both its WE-Hemi. B and AE-Hemi. B fractions (6.60 and 3.19 wt %, respectively). The bound phenolic content in WB was higher (1160–2848.88 μg GAE/g) than free phenolic content (564.17–724.72 μg GAE/g). The total antioxidant activity (TAA) of WB varied from 11.76 to 13.01 μmol TE/g. The bound protocatechuic and ferulic acids were the major phenolic acids with the highest content in WB of HS-490 and HD-2967 variety, respectively. The major flavonoid identified in WB was bound catechin (70.98–198.58 μg/g) with the highest content in HD-2967. The ferulic acid was quantified as the major phenolic acid in Hemi. B fractions with a significantly higher level in WE-Hem-B (439.0–627.0 μg/g) compared to AE-Hemi. B (5.7–13.0 μg/g) fractions. The level of syringaldehyde varied from 0.8 to 1.4 μg/g in AE-Hemi. B while p-coumaric acid content in AE-Hemi. B and WE-Hemi. B varied from 0.2 to 0.3 and 5.8–9.7 μg/g, respectively. Sinapic acid and syringaldehyde were detected only in WE-Hemi. B fractions of HD-2967 and C-306 varieties. The X-ray diffractograms of WE-Hemi. B and AE-Hemi. B showed an amorphous profile with a broad peak in the region of 2θ 15–23°. The structural elucidation of Hemi. B by 1H NMR analysis indicated the xylose backbone with substitution mainly at C(O)-3 and/or C(O)-2 positions by arabinose residues and presence of esterified ferulic acid residues attached to arabinose side chains.

Arabinogalactan Utilization by Bifidobacterium longum subsp. longum NCC 2705 and Bacteroides caccae ATCC 43185 in Monoculture and Coculture.

Arabinogalactan (AG) has been studied as a potential prebiotic in view of stimulating bifidobacteria presence in the gut microbiota. However, bifidobacteria prefer fermentation of oligosaccharides to that of polysaccharides. The contribution of other gut bacteria may allow better growth of bifidobacteria on AG. β-galactanases and β-galactosidases are the main enzymes for the degradation of AG. Additional enzymes such as α-L-arabinofuranosidase and β-L-arabinopyranosidase are required to remove the arabinose side chains. All of these predicted functions are encoded by the genomes of both Bifidobacterium longum subsp. longum NCC 2705 and Bacteroides caccae ATCC 43185. However, neither strain was able to grow significantly on AG, with 25% (B. longum subsp. longum NCC 2705) and 39% (Bac. caccae ATCC 43185) of AG degraded after 48-h fermentation, respectively. In this study, the β-galactanase, β-galactosidase, α-L-arabinofuranosidase, and β-L-arabinopyranosidase from both strains were investigated. The extracellular β-galactosidases of both B. longum subsp. longum NCC 2705 and Bac. caccae ATCC 43185 were able to cleave the β-1,3; 1,4 and 1,6 linkages. However, the β-galactosidase activity of B. longum subsp. longum NCC 2705 was weaker for the β-1,4 linkage, compared with the β-1,3 and 1,6 linkages. The arabinose side chains of AG inhibited the cleavage of β-1,3 and 1,6 linkages by the endo-β-galactanase from both strains, and partially inhibited the cleavage of β-1,4 linkages by the endo-β-1,4 galactanase from Bac. caccae ATCC 43185. The α-L-arabinofuranosidase and β-L-arabinopyranosidase from both strains were unable to cleave arabinose from AG under the conditions used. These results show limited breakdown of AG by these two strains in monoculture. When cocultured with Bac. caccae ATCC 43185, B. longum subsp. longum NCC 2705 grew significantly better than in monoculture on AG after 6 h of fermentation (p < 0.05). The coculture showed 48% AG degradation after 48 h of fermentation, along with reduced pH. Furthermore, compared to monoculture of Bac. caccae ATCC 43185, the concentration of succinate significantly increased from 0.01 ± 0.01 to 4.41 ± 0.61 mM, whereas propionate significantly decreased from 13.07 ± 0.37 to 9.75 ± 2.01 mM in the coculture (p < 0.05). These results suggest that the growth and metabolic activities of Bac. caccae ATCC 43185 were restrained in the coculture, as the pH decreased due to the metabolism of B. longum subsp. longum NCC 2705.

Gelling mechanism of RG-I enriched citrus pectin: Role of arabinose side-chains in cation- and acid-induced gelation

RG-I enriched pectin is present in fruit and vegetable containing products. However, it is removed by the hot acid treatment during commercial pectin production to improve gelling properties and to afford a more uniform pectin quality. Recently, an awareness of the health benefits of RG-I enriched pectin has caused technologists to rethink its utilization by the food industry, especially as a novel healthy gelling agent. Unique RG-I enriched pectin with abundant arabinan side-chains was extracted from citrus membrane by sequential mild acidic and alkaline treatment. Arabinose was then removed by enzymatic treatment to investigate the impact of arabinose side-chains on gelation. The properties of RG-I enriched pectin gels, prepared using cations or acid, showed it could form gels under conditions required for both low and high methoxyl pectin as a result of its highly branched structure. In cation-induced gelation, the HG region forms egg-box junction zones with divalent cations and the side-chains of the RG-I region stabilizes the network structure through entanglements. In acid-induced gelation, low pH promotes formation of hydrogen bonding and hydrophobic interactions within the HG region and the side-chains create a tighter conformation, eventually allowing for stronger interactions between the pectin chains.

Structural characterisation of polysaccharides from roasted hazelnut skins

Two polysaccharide fractions sequentially extracted with water 1W and alkali 1A, were isolated from the hazelnut skins. The monosaccharide composition together with the FTIR and NMR analyses, indicated that both fractions are formed from a mixture of polysaccharides. The fraction 1W consists of methyl-esterified pectic polysaccharide with rhamnogalacturonan I blocks, branching with arabinose side chains, and with 1,5-, 1,3,5-arabinan and galactan polysaccharides. The fraction 1A is a mixture of deesterified rhamnogalacturonan I and 1,5-, 1,3,5-arabinan and 4-O-Me-glucuronoxylan polysaccharides. The presence of unsaturated galacturonic acid and the heterogeneity of the molecular weights, which Mw ranged between 3.6 and 39 kg mol−1, indicated the pectin degradation during roasting.

The modular arabinanolytic enzyme Abf43A-Abf43B-Abf43C from Ruminiclostridium josui consists of three GH43 modules classified in different subfamilies

The abnA gene from Ruminiclostridium josui encodes the large modular arabinanolytic enzyme, Abf43A-Abf43B-Abf43C, consisting of an N-terminal signal peptide, a Laminin_G_3 module, a GH43_22 module, a Laminin_G_3 module, a Big_4 module, a GH43_26 module, a GH43_34 module and a dockerin module in order with a calculated molecular weight of 204,108. Three truncated enzymes were recombinantly produced in Escherichia coli and biochemically characterized, RjAbf43A consisting of the first Laminin_G_3 module and GH43_22 module, RjAbf43B consisting of the second Laminin_G_3 module, Big_4 module and GH43_26 module, and RjAbf43C consisting of the GH43_34 module. RjAbf43A showed a strong α-l-arabinofuranosidase activity toward sugar beet arabinan, highly branched arabinan but not linear arabinan, thus it acted in the removal of arabinose side chains from sugar beet arabinan. By contrast, RjAbf43B showed a strong exo-α-1,5-l-arabinofuranosidase activity toward linear arabinan and arabinooligosaccharides whereas RjAbf43C showed low activity toward these substrates. Although RjAbf43B was activated by the presence of some metal ions such as Zn2+, Mg2+ and Ni2+, RjAbf43A was inhibited by these ions. RjAbf43A and RjAbf43B attacked sugar beet arabinan in a synergistic manner. By comparison, RjAbf43A-Abf43B containing both GH43_22 and GH43_26 modules showed lower hydrolytic activity toward sugar beet arabinan but higher activity toward sugar beet fiber than the sum of the individual activities of RjAbf43A and RjAbf43B, suggesting that the coexistence of two distinct GH43 modules in a single polypeptide is important for the efficient hydrolysis of an insoluble and natural polysaccharide but not a soluble substrate.

UDP-arabinopyranose mutase gene expressions are required for the biosynthesis of the arabinose side chain of both pectin and arabinoxyloglucan, and normal leaf expansion in Nicotiana tabacum.

Plant cell walls are composed of polysaccharides such as cellulose, hemicelluloses, and pectins, whose location and function differ depending on plant type. Arabinose is a constituent of many different cell wall components, including pectic rhamnogalacturonan I (RG-I) and II (RG-II), glucuronoarabinoxylans (GAX), and arabinoxyloglucan (AXG). Arabinose is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. The UDP-arabinopyranose mutases (UAMs) have been demonstrated to convert UDP-arabinopyranose (UDP-Arap) to UDP-arabinofuranose (UDP-Araf) in rice (Oryza sativa L.). The UAMs have been implicated in polysaccharide biosynthesis and developmental processes. Arabinose residues could be a component of many polysaccharides, including branched (1→5)-α-arabinans, arabinogalactans in pectic polysaccharides, and arabinoxyloglucans, which are abundant in the cell walls of solanaceous plants. Therefore, to elucidate the role of UAMs and arabinan side chains, we analyzed the UAM RNA interference transformants in tobacco (Nicotiana tabacum L.). The tobacco UAM gene family consists of four members. We generated RNAi transformants (NtUAM-KD) to down-regulate all four of the UAM members. The NtUAM-KD showed abnormal leaf development in the form of a callus-like structure and many holes in the leaf epidermis. A clear reduction in the pectic arabinan content was observed in the tissue of the NtUAM-KD leaf. The arabinose/xylose ratio in the xyloglucan-rich cell wall fraction was drastically reduced in NtUAM-KD. These results suggest that UAMs are required for Ara side chain biosynthesis in both RG-I and AXG in Solanaceae plants, and that arabinan-mediated cell wall networks might be important for normal leaf expansion.

Biochemical and Structural Analyses of Two Cryptic Esterases in Bacteroides intestinalis and their Synergistic Activities with Cognate Xylanases

Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s−1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases.

Molecular determinants of substrate specificity revealed by the structure of Clostridium thermocellum arabinofuranosidase 43A from glycosyl hydrolase family 43 subfamily 16.

The recent division of the large glycoside hydrolase family 43 (GH43) into subfamilies offers a renewed opportunity to develop structure-function studies aimed at clarifying the molecular determinants of substrate specificity in carbohydrate-degrading enzymes. α-L-Arabinofuranosidases (EC 3.2.1.55) remove arabinose side chains from heteropolysaccharides such as xylan and arabinan. However, there is some evidence suggesting that arabinofuranosidases are substrate-specific, being unable to display a debranching activity on different polysaccharides. Here, the structure of Clostridium thermocellum arabinofuranosidase 43A (CtAbf43A), which has been shown to act in the removal of arabinose side chains from arabinoxylan but not from pectic arabinan, is reported. CtAbf43A belongs to GH43 subfamily 16, the members of which have a restricted capacity to attack xylans. The crystal structure of CtAbf43A comprises a five-bladed β-propeller fold typical of GH43 enzymes. CtAbf43A displays a highly compact architecture compatible with its high thermostability. Analysis of CtAbf43A along with the other member of GH43 subfamily 16 with known structure, the Bacillus subtilis arabinofuranosidase BsAXH-m2,3, suggests that the specificity of subfamily 16 for arabinoxylan is conferred by a long surface substrate-binding cleft that is complementary to the xylan backbone. The lack of a curved-shaped carbohydrate-interacting platform precludes GH43 subfamily 16 enzymes from interacting with the nonlinear arabinan scaffold and therefore from deconstructing this polysaccharide.

Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans.

Background: Switchgrass (Panicum virgatum L.) is a C4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall’s natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf.Results: The expression of a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control.Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.