Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Alebeek, Gert-Jan W. M. van; Zabotina, Olga A.; Beldman, G.; Schols, Henk A.; Voragen, A.G.J.

doi:10.1002/1096-9888(200007)35:7<831::aid-jms7>3.0.co;2-4

Cited by 23 publications

(5 citation statements)

References 27 publications

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“…⌬4,5 Unsaturated oligoGalpA were purified from a saponified pectin lyase digest of a 1% (w/v) 70% DM pectin solution. The oligoGalpA were separated on a source Q column and isolated as described before (12,15). The purity of the oligoGalpA (DP2-10) was determined using HPAEC, pH 12, equipped with PAD detection (12) and found to be in the range of 90 -99% based on area percentages.…”

Section: Methodsmentioning

confidence: 93%

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Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Alebeek¹,

Christensen²,

Schols³

et al. 2002

Journal of Biological Chemistry

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A thorough investigation of the mode of action of Aspergillus niger (4M-147) pectin lyase A (PLA) on differently C 6 -substituted oligogalacturonides is described. PLA appeared to be very specific for fully methyl-esterified oligogalacturonides: removal of the methyl-ester or changing the type of ester (ethyl esterification) or transamidation resulted in (almost) complete loss of conversion. The PLA activity increased with increasing length of the substrate up to a degree of polymerization (DP) of 8 indicating the presence of at least eight subsites on the enzyme. Product analysis demonstrated the formation of several ⌬4,5 unsaturated products and their saturated counterparts. The ⌬4,5 unsaturated trimer was the main product up to DP 8. For DP 9 and 10 ⌬4,5 unsaturated tetramer was the major product. Based upon the bond cleavage frequencies, a provisional subsite map was calculated, which supports the presence of eight subsites. By limited alkaline de-esterification of fully methyl-esterified pentamer and hexamer two sets of partially methyl-esterified pentamers (x and y methyl groups) and hexamers (a and b methyl groups) were prepared. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS) analysis demonstrated that the methyl-ester distribution was fully random. Using these partially methyl-esterified oligogalacturonides as substrates for PLA a 10-fold decrease in reaction rate was recorded compared with the fully methyl-esterified counterparts. Analysis of the methyl-ester distribution of the products showed that PLA tolerates carboxyl groups in the substrate binding cleft. At either subsite ؉2, ؉4, or ؊1 to ؊4 a free carboxyl group could be tolerated, whereas methyl-esters were obligatory at subsite ؉1 and ؉3. So PLA is capable to cleave the bond between a methyl-esterified and a nonesterified galacturonic acid residue, where the newly formed ⌬4,5 unsaturated non-reducing end residue always contains a methyl-ester.Pectin is found in the middle lamella of the primary cell wall of practically all higher plant tissues (1, 2). It is composed of a homogalacturonan backbone (smooth region) interrupted by heavily branched regions of rhamnogalacturonan (hairy region). Homogalacturonan consists of ␣-134-linked D-galacturonic acid (GalpA) 1 residues, which can be methyl-esterified (1-4).Both pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2) are able to depolymerize the smooth region of pectin by ␤-elimination resulting in the formation of a ⌬4,5 unsaturated bond at the newly formed non-reducing end. Although pectate lyase cleaves between two ␣-1,4-D-GalpA residues and pectin lyase cleaves between two methyl-esterified ␣-1,4-D-GalpA residues, this distinction on substrate specificity is not strict. Pectate lyase is generally active on pectin with a moderate degree of methyl esterification (DM) (0 -50%), whereas pectin lyase activity often increases with increasing DM. The only discriminating property is that pectate lyases characterized to date need Ca 2ϩ ions for their acti...

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Section: Methodsmentioning

confidence: 93%

“…1E) were made (13). Here we report the activity of PLA on the various substrates and the mode of action on those substrates where significant activity was observed carried out by newly developed techniques like high performance anion-exchange chromatography (HPAEC) and mass spectrometry (14,15).…”

mentioning

confidence: 99%

Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Alebeek¹,

Christensen²,

Schols³

et al. 2002

Journal of Biological Chemistry

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“…Matrix assisted laser desorption/ionisation MS is often used for offline MS analysis, due to its tolerance to residual salts, the relative simple sample preparation, and high speed of analysis [136]. Using this technique, masses of oligosaccharides and their MS-fragmentation products can be determined [140]. Iontrap MS is used as an alternative to gain more detailed structural information of a specific compound, through multiple MS analysis stages [141].…”

Section: Analytical Approachesmentioning

confidence: 99%

Pectin, a versatile polysaccharide present in plant cell walls

et al. 2009

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Pectin or pectic substances are collective names for a group of closely associated polysaccharides present in plant cell walls where they contribute to complex physiological processes like cell growth and cell differentiation and so determine the integrity and rigidity of plant tissue. They also play an important role in the defence mechanisms against plant pathogens and wounding. As constituents of plant cell walls and due to their anionic nature, pectic polysaccharides are considered to be involved in the regulation of ion transport, the porosity of the walls and in this way in the control of the permeability of the walls for enzymes. They also determine the water holding capacity. The amount and composition of pectic molecules in fruits and vegetables and other plant produce strongly determine quality parameters of fresh and processed food products. Pectin is also extracted from suitable agro-by-products like citrus peel and apple pomace and used in the food industry as natural ingredients for their gelling, thickening, and stabilizing properties. Some pectins gain more and more interest for their health modulating activities. Endogenous as well as exogenous enzymes play an important role in determining the pectic structures present in plant tissue, food products, or ingredients at a given time. In this paper functional and structural characteristics of pectin are described with special emphasis on the structural elements making up the pectin molecule, their interconnections and present models which envisage the accommodation of all structural elements in a macromolecule. Attention is also given to analytical methods to study the pectin structure including the use of enzymes as analytical tools. PectinPectin is one of the major plant cell wall components and probably the most complex macromolecule in nature, as it can be composed out of as many as 17 different monosaccharides containing more than 20 different linkages [1][2][3]. Plant functionality of pectinIn a plant, pectin is present in the middle lamella, primary cell and secondary walls and is deposited in the early stages of growth during cell expansion [4]. Its functionality to a plant is quite divers. First, pectin plays an important role in the formation of higher plant cell walls [5], which lend strength and support to a plant and yet are very dynamic structures [4]. In general, the polymeric composition of primary cell walls in dicotyledonous plants consists of approximately 35% pectin, 30% cellulose, 30% hemicellulose, and 5% protein [5]. Grasses contain 2-10% pectin and wood tissue ca 5%. In cell walls of some fruits and vegetables, the pectin content can be substantially higher and the protein content lower [6]. Second, pectin influences various cell wall properties such as porosity, surface charge, pH, and ion balance and therefore is of importance to the ion transport in the cell wall [7]. Furthermore, pectin oligosaccharides are known to activate plant defense

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“…The loeation of de-esterifieation ean not be eontrolled, thus in theory all isomers ean be present. However, during esterifieation it appeared that the redueing and non-redueing end are preferentially esterified [20], therefore it is assumed that the ehemieal deesterifieation is in reverse order eompared to the ehemieal esterifieation and therefore only the isomers with one earboxyl-group of the inner GalpA-residues are most likely present. Incubation of PLA with these substrates had two effeets.…”

Section: Mode Of Action Of a Niger Pectin Lyase Amentioning

confidence: 99%

Mode of Action Analysis and Structure-Function Relationships of Aspergillus Niger Pectinolytic Enzymes

Benen¹,

Alebeek²,

Voragen³

et al. 2003

Advances in Pectin and Pectinase Research

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As a result of the cloning and overexpression of individual Aspergillus niger pectinase genes the corresponding enzymes are now readily available for detailed characterisation using defmed substrates, including derivatised oligogalacturonides. In this chapter we give an overview of the results of these studies for a pectate and pectine lyase and a polygalacturonase. Detailed kinetic studies on pectate lyase have revealed that the substrate binds to the enzyme as a Ca 2 +-substrate complex, thus explaining the absolute requirement of pectate lyases for Ca 2 +-ions. Using derivatised oligogalacturonides it was shown that pectin lyase A cannot cleave substrates that are: 1) fully de-esterified, 2) fully ethyl-esterified, 3) fully amidated, and 4) fully methylamidated. Furthermore, partial de-esterification of fully methyl esterified oligogalacturonides, one methyl group on average, resulted in a strong reduction of the catalytic efficiency and a change in the preferred binding mode. From these studies it can be concluded that pectin lyase A is highly specific for pectin with a high degree of methylesterification. Endopolygalacturonases (endoPGs) were thouroughly characterised kinetically which allowed to ca1culate subsite affinities. Site directed mutagenesis of endoPGII in combination with the 3D-structure allowed to pin-point amino acids involved in catalysis as well as amino acids that are important for binding of the substrate. At subsite -5 the presence of an arginine appeared critical for displaying multiple attack attack on a single chain (processive) behavior.

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Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cited by 23 publications

References 27 publications

Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Mode of Action of Pectin Lyase A of Aspergillus nigeron Differently C6-substituted Oligogalacturonides

Pectin, a versatile polysaccharide present in plant cell walls

Mode of Action Analysis and Structure-Function Relationships of Aspergillus Niger Pectinolytic Enzymes

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