[Show abstract][Hide abstract] ABSTRACT: Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post- translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood.
We have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses beta-1,6-galactosyltransferase activity, elongating beta-1,6-galactan side chains and forming 6-Gal branches on the beta-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by Forster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of beta-1,6-galactosyltransferase activities compared to AtGALT29A alone.
AtGALT29A is a beta-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to beta-1,6-galactan and to beta-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes.
[Show abstract][Hide abstract] ABSTRACT: α-L-Arabinofuranosidase belongs to the glycoside hydrolase family 62 (GH62) hydrolyze arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-L-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-L-arabinofuranosidase from Streptomyces coelicolor, which comprises a carbohydrate binding module family 13 domain at its N-terminus and a catalytic domain at its C-terminus. The catalytic domain was a five-bladed β-propeller comprising five radially oriented anti-parallel β-sheets. Sugar complex structures with L-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an L-arabinose binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp202 and Glu361 residues were catalytic residues, and Trp270, Tyr461, and Asn462 residues were involved in the substrate binding site for discriminating substrate structures. In particular, hydrogen bonding between Asn462 and xylose at the +2 nonreducing end subsite was important for higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.
Journal of Biological Chemistry 01/2014; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml(-1) protein with 30.5% 3-methyl-1,5-pentanediol in 50 mM tris-HCl pH 8.0) with a 1000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm(3) volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.
[Show abstract][Hide abstract] ABSTRACT: To improve the pentose fermentation rate in Flammulina velutipes, the putative xylose isomerase (XI) gene from Arabidopsis thaliana was cloned and introduced into F. velutipes and the gene expression was evaluated in transformants. mRNA expression of the putative XI gene and XI activity were observed in two transformants, indicating that the putative gene from A. thaliana was successfully expressed in F. velutipes as a xylose isomerase. In addition, ethanol production from xylose was increased in the recombinant strains. This is the first report demonstrating the possibility of using plant genes as candidates for improving the characteristics of F. velutipes.
[Show abstract][Hide abstract] ABSTRACT: ArabinoGalactan Proteins (AGPs) are a complex family of cell wall proteoglycans, which are thought to have major roles in plant growth and development. Genetic approaches studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for the genetic dissection of the role of AGPs in development by modifying their glycan sidechains. We have identified an Arabidopsis glycosyltransferase of the CAZY family GT31 (AtGALT31A), which galactosylates AGP sidechains. A mutation in the AtGALT31A gene causes the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular stage embryos is consistent with a role for AtGALT31A in the progression of embryo development beyond the globular stage. The first observable defect in the mutant is the perturbation of the formative asymmetric division of the hypophysis indicating an essential role for AGP proteoglycans in either the specification of the hypophysis or in the orientation of the asymmetric division plane. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: α-L-Rhamnosidases hydrolyze α-linked L-rhamnosides from oligosaccharides or polysaccharides. We determined the crystal structure of the glycoside hydrolase family 78 Streptomyces avermitilis α-L-rhamnosidase (SaRha78A) in its free and L-rhamnose complexed forms, which revealed the presence of six domains N, D, E, F A and C. In the ligand complex, L-rhamnose was bound in the proposed active site of the catalytic module, revealing the likely catalytic mechanism of SaRha78A. Glu-636 is predicted to donate protons to the glycosidic oxygen, and Glu-895 is the likely catalytic general base, activating the nucleophilic water, indicating that the enzyme operates through an inverting mechanism. Replacement of Glu-636 and Glu-895 resulted in significant loss of α-rhamnosidase activity. Domain D also bound L-rhamnose in a calcium-dependent manner, with a KD of 135 μM. Domain D is thus a non-catalytic carbohydrate binding module (designated SaCBM67). Mutagenesis and structural data identified the amino acids in SaCBM67 that target the features of L-rhamnose that distinguishes it from the other major sugars present in plant cell walls. Inactivation of SaCBM67 caused a substantial reduction in the activity of SaRha78A against the polysaccharide composite gum arabic, but not against aryl rhamnosides, indicating that SaCBM67 contributes to enzyme function against insoluble substrates.
Journal of Biological Chemistry 03/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40-60 g l(-1) ethanol from 15 % (w/v) d-glucose, d-fructose, d-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83 % observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15 % (w/v) SCBC with commercial cellulase was approximately 20 %. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15 % SCBC with the production of β-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g(-1) biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6 %. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.
[Show abstract][Hide abstract] ABSTRACT: The putative α-L-rhamnosidase gene from Streptomyces avermitilis was cloned and expressed. The recombinant enzyme released L-rhamnose from p-nitrophenyl α-L-rhamnoside, Citrus flavonoids such as naringin, rutin, and hesperidin, and gum arabic which is an arabinogalactan-protein. Calcium ions increased L-rhamnose production by the enzyme from gum arabic, whereas enzyme activity was not affected by any metal ions.
Bioscience Biotechnology and Biochemistry 01/2013; · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yariv phenylglycosides, 1,3,5-tri-(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene, are a group of chemical compounds, which selectively bind to arabinogalactan-proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs, as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has so far not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish root AGP was not reduced after enzyme treatment to remove alpha-L-arabinofuranosyl and beta-glucuronosyl residues and beta-1,6-galactan side chains, it was completely lost after degradation of the beta-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia AGPs, increased rather than decreased during the repeated degradation of beta-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, beta-1,3-galactooligosaccharides longer than beta-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides crosslinked to hydrazine beads detected interaction of beta-1,3-galactooligosaccharides longer than beta-1,3-galactopentaose with Yariv phenylglycoside. To the contrary no interactaction with Yariv was detected for beta-1,6-galactooligosaccharides of any length. Therefore we conclude that Yariv phenylglycosides should be considered specific binding reagents for beta-1,3-galactan chains longer than five residues, and seven residues are sufficient for crosslinking, leading to precipitation of the Yariv phenylglycosides.
[Show abstract][Hide abstract] ABSTRACT: We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to β-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.
Applied and Environmental Microbiology 08/2012; 78(22):7939-45. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Substrate specificity is one of the most important functional property of enzymes. We use family 10 xylanase from Streptomyces olivaceoviridis as a model for substrate specificity of glycoside hydrolases. Seven variants were initially designed to change the preference from xylose to glucose at substrate binding subsites -2 and -1. The known mobility of Trp at the -1 subsite and the influence of its environment, which is different in subset 1 and subset 2 family 10 enzymes, were taken into account in variant design. Q88A/R275A had the best ratio of p-nitrophenyl cellobioside vs p-nitrophenyl xylobioside hydrolyzing activity in the first series of variants. The crystal structure shows a movement of Trp274 compared to the native, as a result of loss of interaction with the long side chain of Arg275. The movement creates extra space for the hydroxymethyl of glucose, resulting in improved K-m on glucose derived substrates, while the negative effect on k(cat) is compensated by the Q88A mutation, which also contributes to a further reduction of K-m. Further mutagenesis based on the Q88A/R275A variant resulted in 5.2 times improvement compared to the wild-type p-nitrophenyl cellobioside hydrolyzing activity, which is the best improvement obtained so far for an engineered xylanase. (C) 2011 Elsevier Ltd. All rights reserved.
PROCESS BIOCHEMISTRY 03/2012; 47(3):358-365. · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.
Journal of Biological Chemistry 02/2012; 287(17):14069-77. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of lime pretreatment of brown midrib sorghums on enzymatic saccharification was investigated. Under most of the pretreatment conditions, the saccharification yields of bmrs were higher than those of the normal counterparts. This result suggests that bmr is useful to reduce pretreatment costs, because the amount of lime necessary for the pretreatment of biomass can reduced by using bmr mutants.
Bioscience Biotechnology and Biochemistry 12/2011; 75(12):2415-7. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.
Journal of Biological Chemistry 06/2011; 286(31):27848-54. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An arabinogalactan-protein (AGP) from whole grain of oat (Avena sativa L.) has been isolated for the first time by double precipitation with β-glucosyl Yariv reagent and characterized with regard to its polysaccharide and protein part. The large carbohydrate moiety is rich in galactose (63.0% w/w) and arabinose (32.8% w/w) and free of uronic acids. Linkage analysis of AGP and its products obtained by partial acid hydrolysis as well as enzymatic degradation with specific recombinant enzymes revealed that the carbohydrate moiety consists of a 1,3-Galp backbone and is linked in position 6 to short 1,6-Galp chains, terminating in Araf. In the protein part, high contents of alanine, hydroxyproline and serine could be found which are typical for AGPs. The molecular mass of AGP was determined to be 83 kDa. The carbohydrate moieties, released by alkaline degradation of the protein part, had a size of about 7 kDa. Consequently, the overall structure of the AGP from oat could be assigned to be consistent with the wattle-blossom model of AGPs.
Journal of Cereal Science 03/2011; 53(2):244-249. · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an
array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism- and structure-based families. Genomic
data has shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43
(GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium,
Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly
exo-acting enzymes to degrade arabinan into arabinose, while B. thetaiotaomicron deploys a combination of endo and side chain-cleaving
glycoside hydrolases. Both organisms, however, utilize an arabinan-specific alpha-1,2-arabinofuranosidase in the degradative
process, an activity that has not previously been reported. The enzyme can cleave alpha-1,2-arabinofuranose decorations in
single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure
of the C. japonicus arabinan-specific alpha-1,2-arabinofuranosidase, CjAbf43A displays a 5-bladed beta-propeller fold. The
specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the
polysaccharide. The specificity of CjAbf43A for alpha-1,2-L-arabinofuranose side chains is conferred by a polar residue that
orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelf-like
structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2
linkages that are components of single or double substitutions.
Journal of Biological Chemistry 02/2011; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.
Journal of Biological Chemistry 02/2011; 286(17):15483-95. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: D-xylose isomerase was cloned and characterized from a newly isolated actinobacteria strain, Thermobifisa fusca MBL 10003. There was only one base difference between the xylose isomerase genes from T. fusca XY, a cellulosic bacteria, and T. fusca MBL10003. The structural gene (xylA) is predicted to encode a polypeptide of 387 amino acids with an estimated molecular mass of 43,900. The deduced amino acid sequence of the gene showed high identity with homologous enzymes from the species of Streptomyces, Actinoplanes and Arthrobacter. The optimal temperature and pH were 75°C and 10, respectively. The enzyme was stable between 70 and 75°C, and between pH 4 and 12. Unlike other known xylose isomerases, the T. fusca enzyme had very similar Km values for xylose and glucose, 264 and 274 mM, respectively. In contrast, kcat and, therefore, kcat/Km for xylose was approximately 18-fold larger than that for glucose. It required divalent metal ions for activity. Of these, Mg2+ was the most effective activator, and Co2+ functioned as a co-activator of Mg2+.
Journal of Applied Glycoscience. 01/2011; 59(1):43-46.
[Show abstract][Hide abstract] ABSTRACT: α-Glucuronidase from Streptomyces pristinaespiralis (SpGlcA115A) is composed of a single-chain peptide containing a catalytic domain belonging to glycosyl hydrolase family 115, a novel family of hemicellulolytic α-glucuronidases. The enzyme catalyzes the hydrolysis of α-linked 4-O-methylglucuronosyl and glucuronosyl residues from both polymeric xylans and oligosaccharides. SpGlcA115A was crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals belonged to space group R3 and diffracted to a resolution of 1.9 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2011; 67(Pt 1):68-71. · 0.57 Impact Factor