Multiple Attack Mechanism Research Articles

Pectin Methylesterases: A Review

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME catalyzes reactions according to the double-displacement mechanism. In plants, PMEs can be classified on the basis of presence or absence of the PRO domain in pectin methylesterase into Type 1 and Type II. Type1 contains one to three PRO domains and two or three introns, type II PMEs are without PRO domain and with five or six introns. Once PMEs are secreted into the cell wall, mature PMEs exhibit three different modes of action: single chain mechanism, multiple chain mechanism and multiple attack mechanism. For bacterial and plant PMEs, single chain and multiple chain mechanism is used and for fungal PMEs only multiple chain mechanism has been proposed. PME activity is regulated by differential expression both spatially and temporally.

Characterization of charged functional domains introduced into a modified pectic homogalacturonan by an acidic plant pectin methylesterase (Ficus awkeotsang Makino) and modeling of enzyme mode of action

An acidic plant pectin methylesterase from Ficus awkeotsang achenes (FaPME) was used to demethylesterify a model homogalacturonan (HG) at pH 4.5 and 7.5. Introduced demethylesterified blocks (DMBs) were released by a limited endo-polygalacturonase (EPG) digestion, separated and quantified by HPAEC. The average DMB size (BS¯) and number of such blocks per molecule (BN¯) differed depending on the degree of methylesterification (DM) and reaction pH (P < 0.05). Significant increases in BS¯ and BN¯ were observed in HGs of 30% DM compared to higher DMs. HGs demethylesterified to 30% and 50% DM at pH 4.5 showed significantly larger BS¯ compared to pH 7.5. Absolute degree of blockiness (DBabs), obtained using exhaustive EPG digestions, displayed a linear relationship with the DM regardless of reaction pH (P < 0.001). The distribution of DMBs released by the limited EPG digest was predicted by mathematical modeling and compared with the experimental results. The in silico modeled enzyme mode of action suggested that a random, multiple chain, non-processive mode of action best explains the distributions of small blocks (BS¯≥11) and a processive multiple attack mechanism best explains the distributions of longer blocks. Decreasing the DM of the HGs by the FaPME increased the G′ and G″ of calcium-mediated gels. Pearson's correlation displayed significant correlation coefficients between BS¯, BN¯, DBabs, DM, and G′. The results suggest the possibility to control BS¯ and to produce a uniform population of demethylesterified pectin molecules, particularly in acidic environments where most basic plant PMEs are less active.

Nanostructural modification of a model homogalacturonan with a novel pectin methylesterase: Effects of pH on nanostructure, enzyme mode of action and substrate functionality

A Carica papaya pectin methylesterase (CpL-PME) present in a commercial papain preparation was used to modify the amount and distribution of anionic charge in a population of a model pectic homogalacturonan (HG) at pH 4.5 and pH 7.5. Introduced negatively charged demethylesterified blocks (DMB) were excised as oligomers with limited endo-polygalacturonase (EPG) digestion, and then separated and quantified by high performance anion exchange chromatography. Exhaustive EPG digestion was also performed and the concentrations of mono-, di- and tri-galacturonic acid (GalA) produced were estimated. The CpL-PME mode of action was modeled while the HG population's nanostructure and the resulting functionality of the modified pectin were characterized. The results indicated that average DMB size and number of these “average” blocks per molecule increased as the overall degree of methylesterification (DM) decreased by CpL-PME treatment (P < 0.05). Average DMB size differed depending on the reaction pH at each DM. Absolute degree of blockiness (DBabs) increased as DM decreased and demonstrated a linear relationship with the DM at both reaction pH values. Significant positive correlations between DBabs and average DMB size were observed for both pH series (P < 0.05). The enzyme mode of action, modeled in silico, demonstrates a processive multiple attack mechanism. Functionally, the storage modulus of ionotropic gels formed from the processed substrates increased as DM decreased, and as DBabs and average DMB size increased with highly significant correlations (P < 0.001). These results indicate it is feasible to engineer pectin nanostructure and functionality with CpL-PME.

Alginate Sequencing: An Analysis of Block Distribution in Alginates Using Specific Alginate Degrading Enzymes

Distribution and proportion of β-D-mannuronic and α-L-guluronic acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diguluronic acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with (1)H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (F(G) = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerases act by a multiple attack mechanism. Alternating sequences (MG-blocks) were found to be much shorter than the other block types. In connection with method development, an oligomer library comprising both saturated and unsaturated oligomers of various composition and DP 2-15 was made.

Enzymatic Modification of a Model Homogalacturonan with the Thermally Tolerant Pectin Methylesterase from Citrus: 1. Nanostructural Characterization, Enzyme Mode of Action, and Effect of pH

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ∼10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).

Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

Germinating barley seeds contain multiple forms of α-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The α-amylases are endo-acting and possess a long substrate binding cleft with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley α-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends on the surface sites and varies for starch granules of different genotypes and botanical origin. The surface sites, moreover, are candidates for being involved in degradation of polysaccharides by a multiple attack mechanism. Future studies of the molecular nature of the multivalent enzyme-substrate interactions will address surface sites in both barley α-amylase 1 and in the related isozyme 2.

Demethylation of a model homogalacturonan with a salt-independent pectin methylesterase from citrus: I. Effect of pH on demethylated block size, block number and enzyme mode of action

A model homogalacturonan (HG) was used to produce a demethylated series by reacting it with a citrus salt-independent pectin methylesterase (PME) at pH 4.5 and 7.5. HGs with a predicted degree of methylesterification (DM) of 90%, 80%, 70%, 60% and 50% were produced. HG structural properties and PME mode of action were probed by performing a limited digest with an endo-polygalacturonase. Liberated demethylated blocks (DMB) were separated and quantified by HPAEC coupled to an evaporative light scattering detector. A significant increase in maximum and average size of released DMBs was observed between 80% and 70% DM at pH 7.5 and between 70% and 60% at pH 4.5. The observed distribution of fragment lengths was compared to theoretical modeled distributions. A multiple-attack mechanism with a degree of processivity ( p) of p = 1 at pH 4.5 and p = 10 at pH 7.5 modeled the observed shorter fragments well, while in all cases the amount of larger fragments detected was more successfully described by a single-chain mechanism.

Kinetic and Structural Analysis of Enzyme Sliding on a Substrate: Multiple Attack in β-Amylase

Beta-amylase (EC 3.2.1.2) is starch-hydrolyzing exo-type enzyme that can catalyze the successive liberation of beta-maltose from the nonreducing ends of alpha-1,4-linked glucopyranosyl polymers. There is a well-known phenomenon called multiple or repetitive attack where the enzyme releases several maltose molecules in a single enzyme-substrate complex. In order to understand it further, we examined the beta-amylase-catalyzed reaction using maltooligosaccharides. The Monte Carlo method was applied for simulation of the beta-amylase-catalyzed reaction including the multiple attack mechanism. Through site-directed mutagenesis, we have successfully prepared a mutant enzyme which may be simulated as a multiple attack action reduced one with retaining significant hydrolytic activity. From the results of X-ray structure analysis of the mutant enzyme, it was clarified that one carboxyl residue plays a very important role in the multiple attack. The multiple attack action needs the force of enzyme sliding on the substrate. In addition, it is important for the multiple attack that the enzyme and substrate have the characteristics of a stable productive substrate-enzyme complex through a hydrogen bond between the nonreducing end of the substrate and the carboxyl residue of the enzyme.

Mode of De-esterification of Alkaline and Acidic Pectin Methyl Esterases at Different pH Conditions

Highly esterified citrus pectin was de-esterified at pH 4.5 and 8.0 by a fungal pectin methyl esterase (PME) that was shown to have an acidic isoelectric pH (pI) and an acidic pH optimum and by a plant PME that was characterized by an alkaline pI and an alkaline pH optimum. Interchain and intrachain de-esterification patterns were studied by digestion of the pectin products with endo-polygalacturonase and subsequent analysis using size exclusion and anion-exchange chromatography. No effect of pH was observed on the de-esterification mode of either of the two enzymes. Acidic, fungal PME converted pectin according to a multiple-chain mechanism, with a limited degree of multiple attack at the intrachain level, both at pH 4.5 and at pH 8.0. A multiple-attack mechanism, with a high degree of multiple attack, was more appropriate to describe the action mode of alkaline, plant PME, both at pH 4.5 and at pH 8.0.

Acetyl xylan esterase-catalyzed deacetylation of chitin and chitosan

Acetyl xylan esterase catalyzes the hydrolysis of N-acetyl groups in chitinous materials of variable degrees of polymerization and acetylation. The influence of substrate accessibility is most notable with substrate of high degree of acetylation (DA). The activity rises sharply as the number of acetyl groups in the substrates decreases and at about 24% DA enzyme activity reaches a maximum. Therefore, based on a multiple-attack mechanism we hypothesize that this maximum represents the ideal acetate microstructure for optimal activity of this enzyme. The enzyme partially deacetylates chitin oligomers DP 2–6 with a plateau in deacetylation observed at DP5. These results show that oligomer length is important for enzyme action. By combining successive alkaline and enzymatic deacetylation a process improvement for production of chitosan is suggested.

Interchain Heterogeneity of Enzymatically Deesterified Lime Pectins

Two series of pectins with different levels and patterns of methyl esterification were produced by treatment of a very highly methylated lime pectin with a fungus- or plant-pectin methylesterase. The interchain distribution of free carboxyl groups was investigated by size exclusion and ion exchange chromatography. "Homogeneous" populations with respect to molar mass or charge density were thereby obtained, and their composition, molar mass, and calcium binding properties were investigated. The composition varies from one size exclusion chromatography fraction to another, the highest molar mass fraction being richer in rhamnogalacturonic sequences and exhibiting a slightly higher degree of methylation (DM). Separation of pectins by ion exchange chromatography revealed a narrow charge density distribution for pectins deesterified by fungus-pectin methylesterase, in agreement with a multichain mechanism. Conversely, pectins deesterified by plant-pectin methylesterase exhibited a very large charge density distribution suggesting a processive mechanism. The interchain polydispersity with regard to DM was however shown to have no impact on calcium binding properties of the different fractions. The progressive dimerization through calcium ions with decreasing DM of pectins deesterified by plant-pectin methylesterase seems to be the result of a peculiar intrachain pattern of methyl esterification that can be attributed to a multiple attack mechanism.

Expression and action pattern of Botryotinia fuckeliana ( Botrytis cinerea) rhamnogalacturonan hydrolase in Pichia pastoris

The cDNA sequence coding for the complete rhamnogalacturonan hydrolase (RGase) of Botryotinia fuckeliana ( Botrytis cinerea) was introduced into Pichia pastoris and expressed under the control of the alcohol oxidase promoter. The RGase was secreted into the medium of the yeast driven by the α-factor secretion peptide and could be purified using the C-terminal His 6-tag fusion. RGase activity was measured using a traditional reducing end assay with linseed rhamnogalacturonan (RG) as the substrate, or with an assay using a fluorescent RG oligomer as the substrate and detection and identification of hydrolysis products by capillary zone electrophoresis (CZE). Both methods showed the recombinant enzyme to have a specific activity of about ten units per milligram of protein. Since the CZE method allows identification of the hydrolysis products, it was used to show that the RGase lacks a multiple attack mechanism and needs at least five GalA-Rha repeating disaccharides to be active. This finding is contrary to the action pattern of the native RGase of Aspergillus aculeatus which has the same substrate length requirement, but exhibits multiple attack, leading to products containing only two and three Rha-GalA repeat units without the appearance of intermediate sized fragments. No plant cell wall degrading enzymes were detected in the culture medium of un-transformed P. pastoris, thus the recombinant enzyme, devoid of extraneous activities, can be applied for fine structural studies on cell walls.

Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5

The mechanism of action of purified apple pectin methylesterase on pectin (degree of methoxylation: DM 75) and methoxylated homogalacturonans (DM 70 and 90) was studied at pH 7.0 (optimal pH of the enzyme) and at pH 4.5 (close to the pH of apple juice). Different interchain distributions of the free carboxyl groups were obtained at pH 7.0 and 4.5: high-performance ion exchange chromatography indicated a typical single chain mechanism at pH 7.0, but a mechanism differing from the single and multiple chain ones at pH 4.5. However, the same intrachain distribution of the newly demethoxylated galacturonic acid residues was observed for both pHs by 1H NMR. The high content of consecutive de-esterified or consecutive esterified galacturonic acid residues suggested that apple PME acted with a multiple attack mechanism on the pectic substrate. The degree of multiple attack of the enzyme was greater than or equal to 10–11.

Monte Carlo simulation of multiple attack mechanism of alpha-amylase.

Porcine pancreatic alpha-amylase (EC 3.2.1.1) produces short maltooligosaccharides from a single enzyme-substrate complex without dissociation by multiple or repetitive attack. Multiple attack is caused by relative sliding of the enzyme along the product chain of the enzyme-product complex without dissociation to form another productive complex. The Monte Carlo method was applied to the multiple attack mechanism to predict product distribution from amylose and amylopectin molecules of arbitrary sizes. The position of the initial attack to make the enzyme-substrate complex and branched reaction paths from the enzyme-product complex were selected by random numbers and probabilities. A simulated product distribution from 100,000 samples of amylose of chain length greater than 80 agreed completely with experimental data at the early stage of hydrolysis of amylose of mean chain length 90. On the other hand, the simulated product distribution from amylopectin agreed with experimental data of potato amylopectin when the effective chain length of the A chain was 9. Since the mean chain length of the A chain of potato amylopectin is 15, it is possible that amylopectin is partially compact in solution, so that the enzyme can recognize and act only on the outer side of the A chain at the early stage of digestion.

Mode of action of chitin deacetylase from Mucor rouxii on partially N-acetylated chitosans

The mode of action of chitin deacetylase (CDA) from Mucor rouxii on fully water–soluble partially N-acetylated chitosans was investigated. The Michaelis–Menten constants of three high-molecular-weight chitosans with initial fraction of acetylated units ( F A) of 0.08, 0.35, and 0.62 were determined to 2.1±0.4, 1.7±0.7, and 2.1±0.3 mg/mL, respectively. The relative rate of enzymatic deacetylation increased linearly with increasing F A on the chitosans, indicating that CDA does not preferentially attack any sequences in the chitosan molecules. A water-soluble and highly N-acetylated chitosan with F A of 0.681, having a Bernoullian distribution of acetylated ( A) and deacetylated ( D) units, and a number-average degree of polymerization ( dp n ) of 30, was selected as substrate for CDA for more detailed studies of the mode of action. The chitosan was enzymatically deacetylated to decreasing F A-values ( F A of 0.582, 0.400, and 0.188), and the nearest neighbour frequencies ( F AA, F AD, F DA and F DD) were determined by NMR spectroscopy, showing that the transition frequencies F AD and F DA were lower than expected from a random (Bernoullian) distribution in the further enzymatically deacetylated chitosans, while F AA and F DD were higher compared to a random distribution. The experimental results were compared with model data, assuming an endo-type mechanism with no preferential attack at any sequences in the chitosan chain. The comparison suggested that CDA hydrolysed acetyl-groups according to a multiple attack mechanism, with a degree of multiple attack of at least three. No deacetylation could be detected at the non-reducing end of the enzymatically deacetylated chitosans.

Crystal Structure of Recombinant Soybean β-Amylase Complexed with β-Cyclodextrin

In order to study the interaction of soybean beta-amylase with substrate, we solved the crystal structure of beta-cyclodextrin-enzyme complex and compared it with that of alpha-cyclodextrin-enzyme complex. The enzyme was expressed in Escherichia coli at a high level as a soluble and catalytically active protein. The purified recombinant enzyme had properties nearly identical to those of native soybean beta-amylase and formed the same crystals as the native enzyme. The crystal structure of recombinant enzyme complexed with beta-cyclodextrin was refined at 2. 07-A resolution with a final crystallographic R value of 15.8% (Rfree = 21.1%). The root mean square deviation in the position of C-alpha atoms between this recombinant enzyme and the native enzyme was 0.22 A. These results indicate that the expression system established here is suitable for studying structure-function relationships of beta-amylase. The conformation of the bound beta-cyclodextrin takes an ellipsoid shape in contrast to the circular shape of the bound alpha-cyclodextrin. The cyclodextrins shared mainly two glucose binding sites, 3 and 4. The glucose residue 4 was slightly shifted from the maltose binding site. This suggests that the binding site of the cyclodextrins is important for its holding of a cleaved substrate, which enables the multiple attack mechanism of beta-amylase.

Monte Carlo simulation of multiple attack mechanism of beta-amylase-catalyzed reaction.

beta-Amylase (EC 3.2.1.2) produces maltose (dimer) from the nonreducing ends of alpha-1,4 glucosidic bonds of substrates like maltooligosaccharides, amylose, and amylopectin. The enzyme releases several maltose molecules from a single enzyme-substrate complex without dissociation by multiple or repetitive attack containing many branching reaction paths. The Monte Carlo method was applied to the simulation of the beta-amylase-catalyzed reaction including the multiple attack mechanism. The simulation starts from a single enzyme molecule and a finite number of substrate molecules. The selection of the substrate by the enzyme and degree of multiple attack proceeds by random numbers produced from a computer. The simulation was carried out until the whole substrate and the intermediate molecules were consumed. The simulated data were compared with experimental data of sweet potato beta-amylase using heptamer, octamer, nanomer, and 11-mer as substrates. The only adjustable parameter for odd-numbered substrates was the probability of multiple attack, while an additional adjustable parameter (a correction factor due to low reactivity of tetramer) was needed for even-numbered substrates.

Action pattern of porcine pancreatic alpha-amylase on three different series of β-maltooligosaccharide glycosides

A technique for the investigation of the action pattern of porcine pancreatic amylase (PPA) has been developed by utilising as model substrates 2-chloro-4-nitrophenyl (CNP) and 4-nitrophenyl (NP) β-glycosides of maltooligosaccharides of dp 4–8 and some NP derivatives modified at the nonreducing end with a 4,6- O-benzylidene (Bnl) group. The action pattern was investigated by the method of product analysis, using an HPLC method. The product pattern and cleavage frequency was very similar in the CNP- and NP-oligomers and showed that the glucopyranose residue could be replaced by the aglycon group. Modification of the nonreducing end of NP glycosides to give a 4,6-O- benzylidene- d-glucopyranosyl group indicated a favourable interaction between the Bnl group and the subsites (− 3) and (− 5) but an unfavourable one with subsite (− 4), which resulted in a clear shift in the product pattern. The results obtained with the digestion of the benzylidene-protected substrates confirm a multiple attack mechanism for PPA.

Functional similarity of sea urchin and mammalian α-amylases

α-Amylase from the digestive tube of sea urchin, Anthocidaris crassispina, was isolated by acetone powder, ammonium sulfate fractionation, starch absorption and ion exchange chromatography with DEAE. The enzymatic properties were compared with those of α-amylase from Strongylocentrotus nudas and porcine pancreatic α-amylase, a representative mammalian α-amylase. The activity was purified about 88 times from the crude extract. Optimum pH of the α-amylase was about 6.9 in 50 mM NaCl and about 5.7 in the absence of NaCl at 30°C. Chloride ions activate amylolytic activity in addition to causing a shift of the optimum pH; the K d was 10.6 mM at 30°C. The action pattern of the α-amylase using amylose as a substrate showed a multiple attack mechanism similar to S. nudas and porcine pancreatic α-amylase. In addition to the above properties, the α-amylase from A. crassispina was activated by several monovalent anions. The qualitative activation spectrum was the same as that of porcine pancreatic α-amylase, chloride ion being the best anion activator for both α-amylases. The specific proteinacious inhibitor for animal α-amylases purified from the kidney bean, Phaseolus vulgaris, strongly inhibited the sea urchin α-amylase. It was suggested that there are close functional and structural similarities around the active site of sea urchin and mammalian α-amylases.

NMR spectroscopy studies of the action pattern of tomato pectinesterase: generation of block structure in pectin by a multiple-attack mechanism

The enzymatic de-esterification of highly exterified poly( d-galacturonate) (degree of esterification, d.e. ≈ 0.92, dp n ≈ 15) was monitored by 1H NMR spectroscopy. The enzymatic reaction resulted in a high content of homogeneous triads (GGG and EEE) demonstrating the production of a sequential structure. At high d.e. values, the enzymatic activity increased in proportion to the content of GE-diads. A multiple-attack mechanism was indicated by a relatively slow decrease of single G's (EGE) in the sequential structure. Production of EGG-triads in great preference to GGE-triads pointed to a degree of multiple-attack greater than the effective number average block length of E-residues, ≈ 7–8, and, interestingly, the residue at the reducing end was de-esterified faster than that at the non-reducing end. These findings support the assumption that the enzyme attacks in alternating sequences (-GE-) and de-esterifies linearly preferentially towards the reducing end. Simulation-computed results are reported to demonstrate the difference between multiple-attack and multiple-chain mechanisms.