Structural and Biochemical Studies Elucidate the Mechanism of Rhamnogalacturonan Lyase from Aspergillus aculeatus

Jensen, Mikael; Otten, H.; Christensen, Ulla; Borchert, Torben V.; Christensen, L.L.H.; Larsen, Sine; Leggio, Leila Lo

doi:10.1016/j.jmb.2010.09.013

Cited by 29 publications

(17 citation statements)

References 38 publications

(45 reference statements)

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“…For treatment with RG-I lyase prepared from Aspergillus aculeatus (Jensen et al, 2010), samples adjusted to equal amounts of total sugar were dissolved in 500 mL of 0.05 M Tris-HCl solution, pH 8, containing 2 mM CaCl 2 and incubated at 21°C for 24 h. For AG-II analysis, samples were adjusted to equal amounts of total Ara and hydrolyzed by arabinogalactan-specific enzymes as described by Tryfona et al (2012). Carbohydrates were derivatized, electrophoretically separated, and subjected to PACE gel scanning and quantification as described by Goubet et al (2002Goubet et al ( , 2009.…”

Section: Enzymatic Hydrolysis and Pacementioning

confidence: 99%

Aspen tension wood fibers contain β-(1→4)-galactans and acidic arabinogalactans retained by cellulose microfibrils in gelatinous walls

et al. 2015

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Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula 3 Populus tremuloides). b-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. b-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, b-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high b-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

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Section: Enzymatic Hydrolysis and Pacementioning

confidence: 99%

Aspen tension wood fibers contain β-(1→4)-galactans and acidic arabinogalactans retained by cellulose microfibrils in gelatinous walls

et al. 2015

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“…For consecutive digestions with different AGP-specific enzymes, samples were dissolved in the respective digestion buffer and were incubated at 37°C for 24 h. The buffer composition was as follows: 20 mM ammonium acetate, pH 4.6, 10 mM ammonium acetate, pH 4.3, containing 50 mM KCl, and 20 mM ammonium acetate, pH 3.5, containing 150 mM NaCl for exo-b-(1→3)-galactanase, b-glucuronidase, and endo-b-(1→6)-galactanase, respectively. For the RG-I lyase (Jensen et al, 2010) treatment experiments, Arabidopsis leaf AIR and leaf AGP extracts were dissolved in 500 mL of 0.05 M Tris-HCl solution, pH 8.0, containing 2 mM CaCl 2 and were incubated at 21°C for 24 h. The derivatization of carbohydrates was performed according to previously developed protocols (Goubet et al, 2002). Carbohydrate electrophoresis and PACE gel scanning and quantification were performed as described by Goubet et al (2002Goubet et al ( , 2009.…”

Section: Enzymatic Hydrolysis Of Agps and Pace Analysismentioning

confidence: 99%

Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

et al. 2012

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Proteins decorated with arabinogalactan (AG) have important roles in cell wall structure and plant development, yet the structure and biosynthesis of this polysaccharide are poorly understood. To facilitate the analysis of biosynthetic mutants, water-extractable arabinogalactan proteins (AGPs) were isolated from the leaves of Arabidopsis (Arabidopsis thaliana) plants and the structure of the AG carbohydrate component was studied. Enzymes able to hydrolyze specifically AG were utilized to release AG oligosaccharides. The released oligosaccharides were characterized by high-energy matrix-assisted laser desorption ionization-collision-induced dissociation mass spectrometry and polysaccharide analysis by carbohydrate gel electrophoresis. The Arabidopsis AG is composed of a b-(1→3)-galactan backbone with b-(1→6)-D-galactan side chains. The b-(1→6)-galactan side chains vary in length from one to over 20 galactosyl residues, and they are partly substituted with single a-(1→3)-Larabinofuranosyl residues. Additionally, a substantial proportion of the b-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via b-(1→6)-linkages. The b-(1→6)-galactan side chains are occasionally substituted with a-L-fucosyl. In the fucose-deficient murus1 mutant, AGPs lack these fucose modifications. This work demonstrates that Arabidopsis mutants in AGP structure can be identified and characterized. The detailed structural elucidation of the AG polysaccharides from the leaves of Arabidopsis is essential for insights into the structure-function relationships of these molecules and will assist studies on their biosynthesis.

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“…Over 80 % of the initial activity remained after 16 h of incubation at pH 4-9. LCMS IT-TOF analysis detected six PcRGL4A peptides (F1: QGLHGPYSMTFSR, F2: [38,39]. The former two Arg residues are within the N-terminal catalytic domain, and the latter two Arg residues are within the C-terminal domain of the enzyme.…”

Section: Physicochemical Characteristics Of Pcrgl4amentioning

confidence: 99%

Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum

et al. 2015

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Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7-8 and at 40°C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the Aspergillus aculeatus RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in Escherichia coli demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.

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Structural and Biochemical Studies Elucidate the Mechanism of Rhamnogalacturonan Lyase from Aspergillus aculeatus

Cited by 29 publications

References 38 publications

Aspen tension wood fibers contain β-(1→4)-galactans and acidic arabinogalactans retained by cellulose microfibrils in gelatinous walls

Aspen tension wood fibers contain β-(1→4)-galactans and acidic arabinogalactans retained by cellulose microfibrils in gelatinous walls

Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum

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