Rhamnogalacturonan Acetylesterase Research Articles

Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site

Most structures of neutral lipases and esterases have been found to adopt the common α/β hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common α/β hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemienzyme was solved by multiple isomorphous replacement and refined at 2.4 Å to a conventional crystallographic R-factor of 17.9 % ( R free 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel β-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the α/β hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel β-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.

Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium

This study deals with the effect of methyl esters, acetyl groups, and neutral sugar side-chains on the gelation properties of sugar beet pectin with Ca 2+. Sugar beet pectin was treated in the presence of Ca 2+ with the enzymes pectin methyl esterase (PE), pectin acetyl esterase (PAE), rhamnogalacturonan acetyl esterase (RGAE), arabinofuranosidase B (AF) and rhamnogalacturonase (RGase) in various combinations. Addition of RGAE plus PE or PAE plus PE to the pectin–Ca 2+ mixture significantly increased the release of acetyl groups and methyl esters, in comparison to the addition of only PE or PAE. This indicates that PE activity is hindered by the presence of acetyl groups both in the ‘smooth’ and in the `hairy’ regions. Also the PAE activity is hindered by the presence of methyl groups in the ‘smooth’ regions. Treatment with PAE plus PE led to a stiffer gel, as determined by the storage modulus ( G′), than treatment with PE alone, while RGAE plus PE did not improve the gel forming properties. Addition of only PAE to the pectin–Ca 2+ mixture did not result in gel formation. A lower stiffness of the gel was found when RGase combined with RGAE and PE were added to the pectin–Ca 2+ mixture, in comparison to treatment with PE alone. Addition of AF plus PE to the pectin–Ca 2+ mixture gave similar rheological effects as treatment with only PE. A fraction representing the ‘smooth’ homogalacturonan regions, which was obtained after treatment of the beet pectin with RGase and subsequent size-exclusion chromatography, was also able to form a gel with Ca 2+ and PE. However, the gel formation was much slower, and the stiffness of the gel was lower than when the parental extract was used. Also with the modified pectin the treatment with PAE plus PE gave an increased stiffness of the gel in comparison to PE alone.

Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases

Background: The complex polysaccharide rhamnogalacturonan constitutes a major part of the hairy region of pectin. It can have different types of carbohydrate sidechains attached to the rhamnose residues in the backbone of alternating rhamnose and galacturonic acid residues; the galacturonic acid residues can be methylated or acetylated. Aspergillus aculeatus produces enzymes that are able to perform a synergistic degradation of rhamnogalacturonan. The deacetylation of the backbone by rhamnogalacturonan acetylesterase (RGAE) is an essential prerequisite for the subsequent action of the enzymes that cleave the glycosidic bonds. Results: The structure of RGAE has been determined at 1.55 Å resolution. RGAE folds into an α/β/α structure. The active site of RGAE is an open cleft containing a serine–histidine–aspartic acid catalytic triad. The position of the three residues relative to the central parallel β sheet and the lack of the nucleophilic elbow motif found in structures possessing the α/β hydrolase fold show that RGAE does not belong to the α/β hydrolase family. Conclusions: Structural and sequence comparisons have revealed that, despite very low sequence similarities, RGAE is related to seven other proteins. They are all members of a new hydrolase family, the SGNH-hydrolase family, which includes the carbohydrate esterase family 12 as a distinct subfamily. The SGNH-hydrolase family is characterised by having four conserved blocks of residues, each with one completely conserved residue; serine, glycine, asparagine and histidine, respectively. Each of the four residues plays a role in the catalytic function.

O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of a rhamnogalacturonan O-acetyl-transferase from potato suspension-cultured cells.

A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [14C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus, acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 degrees C, an apparent Km of 35 microM and an apparent Vmax of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass > 500 kDa.

Enzymatic degradation of plant cell wall polysaccharides: the kinetic effect of competitive adsorption.

Insoluble potato dietary fibre, isolated from potato pulp, can be enzymatically hydrolysed with the pectolytic enzyme preparation Pectinex Ultra SP from Novo Nordisk A/S, in order to produce soluble fibre. The soluble fibre has valuable functional properties for the food industry. Cloned monocomponent enzymes from Pectinex Ultra SP (arabinofuranosidase, endoglucanase II, pectin lyase, polygalacturonase I, rhamnogalacturonan acetyl esterase, rhamnogalacturonase a, rhamnogalacturonase b and xylanase I) were added in order to increase the yield. Surprisingly, however, the yield is not increased when any of the monocomponent enzymes are added. To describe the results a new model designated 'the competitive activity adsorption model' is proposed. The model is based on the fact that the enzymes are adsorbed to the substrate before action. A combination of the Langmuir adsorption isotherm and basic enzyme kinetics shows that different enzymes that adsorb competitively will have an inhibitory effect on each other and consequently decrease the hydrolysis rate and thereby the yield. The model has been confirmed by an experiment in which the fibre has been pre-treated with rhamnogalacturonan acetyl esterase.

Crystallization and preliminary X-ray diffraction studies of the heterogeneously glycosylated enzyme rhamnogalacturonan acetylesterase from Aspergillus aculeatus.

Well diffracting crystals of rhamnogalacturonan acetylesterase from Aspergillus aculeatus have been obtained in two polymorphic modifications despite its heterogeneous glycosylation. The best-diffracting crystals (resolution 1.55 A) are orthorhombic. The limit of the diffraction pattern of the other (trigonal) form is 2.5 A. The ability of the enzyme to crystallize appears to depend on the glycosylation of the protein sample. This aspect has been investigated by mass spectrometry, which also showed that the orthorhombic crystals have the same glycosylation as the protein sample used in the crystallization.

Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of paeY expression appears to be dependent on the KdgR repressor, which controls all the steps of pectin catabolism, and on the catabolite regulatory protein (CRP), the global activator of sugar catabolism. The contiguous pelD, paeY and pemA genes are transcribed as an operon from a promoter proximal to pelD which allows the regulation by KdgR and CRP. However, transcription can be interrupted at the intra-operon Rho-independent terminator situated between pelD and paeY. The paeY mutant inoculated into Saintpaulia plants was less invasive than the wild-type E. chrysanthemi strain 3937, demonstrating the important role of PaeY in the soft-rot disease.

The relation between different polymorphic modifications of rhamnogalacturonan acetylesterase

The relation between different polymorphic modifications of rhamnogalacturonan acetylesterase

A xylogalacturonan subunit present in the modified hairy regions of apple pectin

The high molecular weight fraction (fraction A) of the modified hairy regions (MHR) from apple cell walls was treated by rhamnogalacturonase (RGase) after saponification and after deacetylation by rhamnogalacturonan acetylesterase (RGAEase). Three fractions could be recognized by size-exclusion chromatography: rhamnogalacturonan oligomers, residual stubs of the rhamnogalacturonan backbone rich in arabinan side-chains, and a fraction rich in xylose and galacturonic acid. The “xylogalacturonans” obtained after saponification and after enzymic deacetylation had rather similar sugar compositions (xylose:galacturonic acid ratios of 0.4–0.9) and molecular weights. After saponification, the xylogalacturonan was eluted as a single peak on anion-exchange chromatography whereas three peaks were obtained when the MHR was deacetylated by RGAEase, indicating variations in the degrees of methylation. One of the methyl-esterified xylogalacturonan fractions was characterized by NMR spectroscopy: the xylose residues were β-(1 → 3)-linked to some of the galacturonic acid residues within a rather high molecular weight xylo-(1 → 4)-α-galacturonan and a degree of methylation of 39 was calculated. The methyl esters were found to be equally divided between the substituted and unsubstituted galacturonosyl residues.

Molecular Cloning and Characterization of a Rhamnogalacturonan Acetylesterase from Aspergillus aculeatus: SYNERGISM BETWEEN RHAMNOGALACTURONAN DEGRADING ENZYMES

A rhamnogalacturonan acetylesterase (RGAE) was purified to homogeneity from the filamentous fungus Aspergillus aculeatus, and the NH2-terminal amino acid sequence was determined. Full-length cDNAs encoding the enzyme were isolated from an A. aculeatus cDNA library using a polymerase chain reaction-generated product as a probe. The 936-base pair rha1 cDNA encodes a 250-residue precursor protein of 26,350 Da, including a 17-amino acid signal peptide. The rha1 cDNA was overexpressed in Aspergillus oryzae, a filamentous fungus that does not possess RGAE activity, and the recombinant enzyme was purified and characterized. Mass spectrometry of the native and recombinant RGAE revealed that the enzymes are heterogeneously glycosylated. In addition, the observed differences in their molecular masses, lectin binding patterns, and monosaccharide compositions indicate that the glycan moieties on the two enzymes are structurally different. The RGAE was shown to act in synergy with rhamnogalacturonase A as well as rhamnogalacturonase B from A. aculeatus in the degradation of apple pectin rhamnogalacturonan. RNA gel blot analyses indicate that the expression of rhamnogalacturonan degrading enzymes by A. acculeatus is regulated at the level of transcription and is subjected to carbon catabolite repression by glucose.

Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectins

Rhamnogalacturonan acetylesterase, able to specifically hydrolyse the acetyl asters present in modified hairy (ramified) regions (MHR) of apple pectin, was identified. The enzyme removed about 70% of the total acetyl groups in MHR. This acetylesterase did not cause the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, including the acetylated smooth regions present in beet pectin. Pretreatment of pectic polysaccharides in order to remove arabinose side chains had no effect on the acetyl release, wor was an effect found on the rate or degree of acetyl release, when the purified acetylesterase was combined with pectolytic enzymes, pectin methylesterase or arabinanases.