Bifunctional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side‐chains of rhamnogalacturonan‐I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP‐Gal to growing β‐1,4‐galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP‐α‐d ‐Gal and the transfer of an arabinopyranose from UDP‐β‐l ‐Ara_p to galactan chains. The two substrates share a similar structure, but UDP‐α‐d ‐Gal is the preferred substrate, with a 10‐fold higher affinity. Transfer of Ara_p to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.     

Organization and nucleotide sequence of the Streptococcus mutans galactose operon

The galactose operon encoding a repressor and genes for the Leloir pathway for galactose metabolism (galactokinase, galactose-1-phosphate-uridyl transferase and UDP glucose-4-epimerase) was located adjacent to the multiple sugar metabolism (msm) operon on the chromosome of Streptococcus mutans Ingbritt (serotype c) and the complete nucleotide sequence of this 5-kilobase region was determined. The Leloir pathway was induced by the presence of galactose in the growth medium or following the release of intracellular galactose after uptake and cleavage of -galactosides by the multiple sugar metabolism system. Analysis of the mechanism of galactose transport confirmed the absence of a galactose-specific phosphotransferase system and suggested the presence of an inducible galactose permease. Evidence is presented that galactose transport is independent of the proton motive force and may be ATP-dependent.     

Crystal structures of the archaeal UDP‐GlcNAc 2‐epimerase from Methanocaldococcus jannaschii reveal a conformational change induced by UDP‐GlcNAc

Uridine diphosphate N ‐ acetylglucosamine (UDP‐GlcNAc) 2‐epimerase catalyzes the interconversion of UDP‐GlcNAc to UDP‐N‐acetylmannosamine (UDP‐ManNAc), which is used in the biosynthesis of cell surface polysaccharides in bacteria. Biochemical experiments have demonstrated that mutation of this enzyme causes changes in cell morphology and the thermoresistance of the cell wall. Here, we present the crystal structures of Methanocaldococcus jannaschii UDP‐GlcNAc 2‐epimerase in open and closed conformations. A comparison of these crystal structures shows that upon UDP and UDP‐GlcNAc binding, the enzyme undergoes conformational changes involving a rigid‐body movement of the C‐terminal domain. We also present the crystal structure of Bacillus subtilis UDP‐GlcNAc 2‐epimerase in the closed conformation in the presence of UDP and UDP‐GlcNAc. Although a structural overlay of these two closed‐form structures reveals that the substrate‐binding site is evolutionarily conserved, some areas of the allosteric site are distinct between the archaeal and bacterial UDP‐GlcNAc 2‐epimerases. This is the first report on the crystal structure of archaeal UDP‐GlcNAc 2‐epimerase, and our results clearly demonstrate the changes between the open and closed conformations of this enzyme. Proteins 2014; 82:1519–1526. © 2014 Wiley Periodicals, Inc.     

Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana

In plants, UDP‐glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP‐sugars required for the biosynthesis of wall matrix polysaccharides. UDP‐glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP‐glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose‐6‐phosphate and glucose‐1‐phosphate, and UDP‐glucose generation by UDP‐glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP‐glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP‐glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP‐glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.     

NDP-sugars, floridoside and floridean starch levels in relation to activities of UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in Solieria chorda l is (Rhodophyceae) under experimental conditions

Under limited nutrient availability (i.e. unenriched sea‐water) and under 75 mol photons m^–2 s^–1 irradiance 12:12 LD, thalli of Solieria chordalis J. Agardh accumulated floridean starch and floridoside. When they were transferred into nutrient‐enriched seawater (150 umol L^?1 NO3¹‐ and 7 umol L^?1 P0₄³i at 35 umol photons m^?2 s^?1 in irradiance 12:12 LD, starch and floridoside levels decreased. The main nucleotide diphosphate (NDP) sugars (i.e. UDP‐glucose, UDP‐galactose and ADP‐glucose) and the activities of UDP‐glucose pyrophosphorylase [Enzyme Code (EC) 2.7.7.9] and UDP‐glucose‐4‐epimerase (EC 5.1.3.2) were measured under these controlled culture conditions. Both UDP‐glucose and UDP‐galactose in the thal l i increased under conditions known to favor the accumulation of floridean starch and floridoside, whereas they decreased under conditions leading to floridean starch and floridoside breakdown. On the other hand, ADP‐glucose level only varied slightly. Although UDP‐glucose pyrophosphorylase activity rose under conditions of floridean starch synthesis, little variation was observed in UDP‐glucose‐4‐epimerase activity. These results suggest a possible enzymatic regulation of the NDP‐sugar and carbohydrate pool in which UDP‐glucose pyrophosphorylase would play a major role.     

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

Nucleotide sugars are considered as bottleneck and expensive substrates for enzymatic glycan synthesis using Leloir‐glycosyltransferases. Synthesis from cheap substrates such as monosaccharides is accomplished by multi‐enzyme cascade reactions. Optimization of product yields in such enzyme modules is dependent on the interplay of multiple parameters of the individual enzymes and governed by a considerable time effort when convential analytic methods like capillary electrophoresis (CE) or HPLC are applied. We here demonstrate for the first time multiplexed CE (MP‐CE) as fast analytical tool for the optimization of nucleotide sugar synthesis with multi‐enzyme cascade reactions. We introduce a universal separation method for nucleotides and nucleotide sugars enabling us to analyze the composition of six different enzyme modules in a high‐throughput format. Optimization of parameters (T, pH, inhibitors, kinetics, cofactors and enzyme amount) employing MP‐CE analysis is demonstrated for enzyme modules for the synthesis of UDP‐α‐D‐glucuronic acid (UDP‐GlcA) and UDP‐α‐D‐galactose (UDP‐Gal). In this way we achieve high space‐time‐yields: 1.8 g/L?h for UDP‐GlcA and 17 g/L?h for UDP‐Gal. The presented MP‐CE methodology has the impact to be used as general analytical tool for fast optimization of multi‐enzyme cascade reactions.     

蛹虫草MF27菌糠多糖对肝细胞氧化损伤的保护作用

蛹虫草是一种可利用大米、小麦等谷物培育的名贵药用真菌,其采收后的菌糠里仍富含许多生物活性物质。本研究立足于蛹虫草菌糠多糖,先分析其化学抗氧化活性,再以H₂O₂诱导氧化应激损伤的LO2细胞为模型,评价其对肝细胞氧化损伤的保护作用,进而解析活性多糖的单糖组分。结果显示,菌糠多糖能够有效清除DPPH自由基、羟基（?OH）自由基和ABTS自由基,EC₅₀分别为0.26mg/mL、1.03mg/mL、0.57mg/mL,提示其具有良好的抗氧化能力;在H₂O₂诱导氧化应激损伤的LO2细胞中,菌糠多糖能有效地保护细胞形态的完整性,并且随浓度梯度递增式地提高细胞存活率,当多糖浓度为5mg/mL时,细胞存活率可达91.83%;在分析其作用机制上,与模型组对比,菌糠多糖能通过调节细胞抗氧化酶SOD（提高4.91倍）和CAT（提高3.40倍）的表达来清除ROS含量（P<0.01）,降低氧化损害;经检测,虫草菌糠活性多糖主要含有葡萄糖、甘露糖、半乳糖、阿拉伯糖、葡萄糖醛酸、木糖、半乳糖醛酸、鼠李糖和岩藻糖等单糖。研究结果表明蛹虫草MF27菌糠多糖具有保护肝细胞氧化损伤的作用,为进一步开发和利用虫草菌糠提供了重要理论依据。     

Cell-Wall Carbohydrates of the Endosperm of the Seed of Gleditsia triacanthos

The endosperm of the seed of Gleditsia triacanthos L. contains 18.55% of its dry weight as nonreserve, cell-wall carbohydrates. Of this carbohydrate material, comprising mainly mannose, galactose, and glucose, 76.1% was of low-molecular weight or highly hydrophilic. Mannose, galactose, and glucose were also the major sugar components of the polysaccharides extracted with alkali (23.1% of the cell-wall), while the same sugars, with minor amounts of arabinose, form the residues. Methylation analysis of the polysaccharides and the borate-sodium hydroxide residue indicate that the cell walls are built up on a network of galactomannans, with high Man/Gal ratios, reinforced with minor amounts of cellulose.     

灵芝不同菌株胞外多糖的单糖组成和抗氧化活性分析

灵芝多糖是药用真菌灵芝的主要活性成分之一。早期研究发现不同灵芝子实体多糖的单糖组成和活性存在差异,但不同灵芝菌株胞外多糖的单糖组成和活性是否有区别仍不清楚。本研究通过液体发酵获得灵芝菌株5.26和5.616的胞外多糖,使用DEAE-cellulose和Sephadex G-200柱色谱分离纯化得到了两种多糖(5.26-2-1和5.616-2-1),并对5.26-2-1,5.616-2-1的单糖组成和抗氧化活性进行了分析。结果表明,5.26-2-1主要由甘露糖、半乳糖醛酸、半乳糖和葡萄糖组成,而5.616-2-1主要由甘露糖、半乳糖和葡萄糖组成。5.26-2-1中葡萄糖的摩尔百分比为63.97%,显著高于5.616-2-1(29.3%),但半乳糖的摩尔百分比为9.34%,显著低于5.616-2-1 (42.78%)。当多糖质量浓度为2 mg/mL时,5.26-2-1的Fe²⁺螯合能力、对1,1-二苯基-2-三硝基苯肼(DPPH)和羟自由基(·OH)的最大清除率分别为71.9%、71.3%和60.8%,显著高于5.616-2-1的值(63.5%、60.4%和51.8%...     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal⁻ ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Role of α-phosphoglucomutase and phosphoglucose isomerase activities at the branching point between sugar catabolism and anabolism in Lactobacillus casei

Aims: To evaluate the role of α‐phosphoglucomutase (α‐Pgm) and phosphoglucose isomerase (Pgi) activities in growth rate, sugar‐phosphates, UDP‐sugars and lactate biosynthesis in Lactobacillus casei. Methods and Results: The pgm and pgi genes coding for α‐Pgm and Pgi activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous overexpression. In MRS fermentation medium with glucose, overexpression of pgm gene in L. casei resulted in a growth rate reduced to 75% and glucose‐6P levels reduced to 47%. By contrast, with lactose, the growth rate was raised to 119%. An increment of α‐Pgm activity had no significant effect on UDP‐sugar levels. Remarkably, Pgi overexpression in L. casei grown in lactose or galactose resulted in almost a double growth rate with respect to the control strain. The increased Pgi activity also resulted in glucose‐6P levels reduced to 25 and 59% of control strain cultured in glucose and lactose, respectively, and the fructose‐6P levels were increased to 128% on glucose. UDP‐glucose and UDP‐galactose levels were reduced to 66 and 55%, respectively, of control strain levels cultured in galactose. In addition, the lactate yield increased to 115% in the strain overproducing Pgi grown in galactose. Conclusions: The physiological amount of α‐Pgm and Pgi activities is limited for L. casei growth on lactose, and lactose and galactose, respectively, and that limitation was overcome by pgm and pgi gene overexpression. The increment of α‐Pgm and Pgi activities, respectively, resulted in modified levels of sugar‐phosphates, sugar‐nucleotides and lactate showing the modulation capacity of the carbon fluxes in L. casei at the level of the glycolytic intermediate glucose‐6P. Significance and Impact of the Study: Knowledge of the role of key enzymes in metabolic fluxes at the branching point between anabolic and catabolic pathways would allow a rational design of engineering strategies in L. casei.     

Isolation of β-galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

β-Galactosidase (EC 3.2.1.23) was purified from the cell wall of the fruit of Japanese pear ( Pyrus serotina Rehder var. culta Rehder cv. Hosui) and characterized. Five peaks of β-galactosidase activity, designated as Gal I to V, were separated by hydrophobic chromatography on butyl toyopearl and ion exchange chromatography on Mono S. These isolated β-galactosidases were investigated with regard to their abilities to release monomeric galactose from the fractionated polymers of native cell wall (cyclo-hexane-trans-1,2-diamine tetraacetic acid-, Na₂CO₃-, guanidine thiocyanate- and KOH-soluble fractions) and arabinogalactan (from larch wood). All the β-galactosidase fractions were active against native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall and arabinogalactan. Gal II released much galactose only from KOH-soluble polymers and arabinogalactan. Gal III released the most galactose. from cyclohexane- trans -1,2-diamine tetraacetic acid-, Na₂CO₃- and guanidine thiocyanate-soluble cell wall polymers which probably contained galactosyl side chains of pectic polymers, although it did not react much to arabinogalactan. In addition, the activity of Gal Ill dramatically increased as ripening proceeded. Furthermore, Gal III was purified to homogeneity by gel filtration on TSKgel 3000SW and the size of a polypeptide was 80 kDa on SDS-PAGE.     

Deficiency of mitochondrial outer membrane protein 64 confers rice resistance to both piercing‐sucking and chewing insects in rice

The brown planthopper (BPH) and striped stem borer (SSB) are the most devastating insect pests in rice (Oryza sativa ) producing areas. Screening for endogenous resistant genes is the most practical strategy for rice insect‐resistance breeding. Forty‐five mutants showing high resistance against BPH were identified in a rice T‐DNA insertion population (11,000 putative homozygous lines) after 4 years of large‐scale field BPH‐resistance phenotype screening. Detailed analysis showed that deficiency of rice mitochondrial outer membrane protein 64 (OM64 ) gene resulted in increased resistance to BPH. Mitochondrial outer membrane protein 64 protein is located in the outer mitochondrial membrane by subcellular localization and its deficiency constitutively activated hydrogen peroxide (H₂O₂) signaling, which stimulated antibiosis and tolerance to BPH. The om64 mutant also showed enhanced resistance to SSB, a chewing insect, which was due to promotion of Jasmonic acid biosynthesis and related responses. Importantly, om64 plants presented no significant changes in rice yield‐related characters. This study confirmed OM64 as a negative regulator of rice herbivore resistance through regulating H₂O₂ production. Mitochondrial outer membrane protein 64 is a potentially efficient candidate to improve BPH and SSB resistance through gene deletion. Why the om64 mutant was resistant to both piercing‐sucking and chewing insects via a gene deficiency in mitochondria is discussed.     

Vitamin B1 THIAMIN REQUIRING1 synthase mediates the maintenance of chloroplast function by regulating sugar and fatty acid metabolism in rice

Vitamin B₁ (VB1), including thiamin, thiamin monophosphate (TMP), and thiamin pyrophosphate (TPP), is an essential micronutrient for all living organisms. Nevertheless, the precise function of VB1 in rice remains unclear. Here, we described a VB1 auxotrophic mutant, chlorotic lethal seedling (cles) from the mutation of OsTH1, which displayed collapsed chloroplast membrane system and decreased pigment content. OsTH1 encoded a phosphomethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase, and was expressed in various tissues, especially in seedlings, leaves, and young panicles. The VB1 content in cles was markedly reduced, despite an increase in the expression of VB1 synthesis genes. The decreased TPP content affected the tricarboxylic acid cycle, pentose phosphate pathway, and de novo fatty acid synthesis, leading to a reduction in fatty acids (C16:0 and C18:0) and sugars (sucrose and glucose) of cles. Additionally, irregular expression of chloroplast membrane synthesis genes led to membrane collapse. We also found that alternative splicing and translation allowed OsTH1 to be localized to both chloroplast and cytosol. Our study revealed that OsTH1 was an essential enzyme in VB1 biosynthesis and played crucial roles in seedling growth and development by participating in fatty acid and sugar metabolism, providing new perspectives on VB1 function in rice.     

The role of arabinokinase in arabinose toxicity in plants

Plant cell wall polymers are synthesized by glycosyltransferases using nucleotide sugars as substrates. Most UDP‐sugars are synthesized from UDP‐glucose via de novo pathways but salvage pathways work in parallel to recycle sugars, which have been released during cell wall polymer and glycoprotein turnover. Here we report on the cloning and biochemical analysis of two arabinokinases in Arabidopsis. Arabinokinase is a 100 kDa protein located in the cytosol with a putative N‐terminal glycosyltransferase domain and a C‐terminal sugar‐1‐kinase domain. This unique structure is highly conserved in the plant kingdom. Arabinokinase has a high affinity for l ‐arabinose, which is the only sugar substrate of this GHMP (galactose; homoserine; mevalonate; phosphomevalonate) kinase. Plants that were knocked‐out for arabinokinase and the previously described ara1‐1 mutant were characterized. The ARA1‐1 mutant form of the enzyme carries a point mutation in an α‐helix. The mutation is close to the substrate binding site and changes the K_m value for arabinose from 80 μm in the wild type to 17 000 μm in ARA1‐1. The previous arabinose toxicity explanation is challenged by knockout plants in arabinokinase that accumulate higher levels of arabinose but do not show signs of arabinose toxicity. Analysis of marker genes from sugar signalling pathways (SnRK1 and Tor) suggest that ara1‐1 misinterprets its carbon energy status. Although glucose is present in ara1‐1 similar to wild type levels, it constitutively changes gene expression as typically found in wild type plants only under starvation conditions. Furthermore, ara1‐1 shows increased expression of marker genes for programmed cell death as found in other lesion mimic mutants.     

In-microbe formation of nucleotide sugars in engineered Escherichia coli

Numerous different nucleotide sugars are used as sugar donors for the biosynthesis of glycans by bacteria, humans, fungi, and plants. However, many of these nucleotide sugars are not available either in their native form or with the sugar portion labeled with a stable or radioactive isotope. Here we demonstrate the use of Escherichia coli metabolically engineered to contain genes that encode proteins that convert monosaccharides into their respective monosaccharide-1-phosphates and subsequently into the corresponding nucleotide sugars. In this system, which we designated “in-microbe”, reactions occur within 2 to 4 h and can be used to generate nucleotide sugars in amounts ranging from 5 to 12.5 μg/ml cell culture. We show that the E. coli can be engineered to produce the seldom observed nucleotide sugars UDP–2-acetamido-2-deoxy-glucuronic acid (UDP–GlcNAcA) and UDP–2-acetamido-2-deoxy-xylose (UDP–XylNAc). Using similar strategies, we also engineered E. coli to synthesize UDP–galacturonic acid (UDP–GalA) and UDP–galactose (UDP–Gal). ¹³C- and ¹⁵N-labeled NDP–sugars are formed using [¹³C] glucose as the carbon source and with [¹⁵N]NH₄Cl as the nitrogen source.     

Secondary sugar binding site identified for LecA lectin from Pseudomonas aeruginosa

The galactose‐specific lectin LecA from Pseudomonas aeruginosa is a target for the development of new anti‐infectious compounds. Sugar based molecules with anti‐adhesive properties present great potential in the fight against bacterial infection and biofilm formation. LecA is specific for oligosaccharides with terminal α‐galactoside residues and displays strong affinity for melibiose (αGal1‐6Glc) with a K_d of 38.8 µM. The crystal structure of LecA/melibiose complex shows classical calcium‐bridged binding of αGal in the primary binding site but also revealed a secondary sugar binding site with glucose bound. This sugar binding site is in close proximity to the galactose binding one, is independent of calcium and mainly involves interactions with a symmetry‐related protein. This discovery would help to the design of new potent inhibitors targeting both binding sites. Proteins 2014; 82:1060–1065. © 2013 Wiley Periodicals, Inc.