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 alpha/beta 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 alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A 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 beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta 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 beta-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.     

Crystal structure of plant pectin methylesterase

Pectin is a principal component in the primary cell wall of plants. During cell development, pectin is modified by pectin methylesterases to give different properties to the cell wall. This report describes the first crystal structure of a plant pectin methylesterase. The beta-helical structure embodies a central cleft, lined by several aromatic residues, that has been deduced to be suitable for pectin binding. The active site is found at the center of this cleft where Asp157 is suggested to act as the nucleophile, Asp136 as an acid/base and Gln113/Gln135 to form an anion hole to stabilize the transition state.     

The peculiar structural features of kiwi fruit pectin methylesterase: amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor

Pectin methylesterase (PME) from kiwi fruit (Actinidia deliciosa) is a glycoprotein, showing an apparent molecular mass of 50 kDa upon size exclusion chromatography and SDS-PAGE. The primary structure, elucidated by direct sequencing of the protein, comprises 321 amino acid residues providing a molecular mass of 35 kDa. The protein has an acetylated Thr residue at the amino terminus and five N-glycosylation consensus sequences, four of which are actually glycosylated. A careful investigation of the oligosaccharide structures demonstrated that PME glycans belong to complex type oligosaccharides essentially consisting of xylosylated polyfucosylated biantennary structures. Alignment with known mature plant PME sequences indicates that the postulated active site residues are conserved. Kiwi PME activity is inhibited following the interaction with the proteinaceous inhibitor PMEI, isolated from the same source. Gel-filtration experiments show that kiwi PME/PMEI complex is stable in a large pH range and dissociates only at pH 10.0. Modeling of the interaction with the inhibitor was performed by using the crystal structure of the complex between kiwi PMEI and tomato PME as a template. The model shows that the binding site is the same reported for tomato PME. However, additional salt link interactions are found to connect the external loops of kiwi PME to PMEI. This finding may explain the higher pH stability of the complex formed by the two kiwi proteins respect to that formed by PMEI and tomato PME.     

Molecular basis of the activity of the phytopathogen pectin methylesterase

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel beta-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.     

Phenylephrine,a small molecule,inhibits pectin methylesterases

Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1?μM. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.     

Pectin methylesterase and its proteinaceous inhibitor: a review

Pectin methylesterase (PME) catalyses the demethoxylation of pectin, a major plant cell wall polysaccharide. Through modification of the number and distribution of methyl-esters on the pectin backbone, PME affects the susceptibility of pectin towards subsequent (non-) enzymatic conversion reactions (e.g., pectin depolymerisation) and gel formation, and, hence, its functionality in both plant cell wall and pectin-containing food products. The enzyme plays a key role in vegetative and reproductive plant development in addition to plant-pathogen interactions. In addition, PME action can impact favourably or deleteriously on the structural quality of plant-derived food products. Consequently, PME and also the proteinaceous PME inhibitor (PMEI) found in several plant species and specifically inhibiting plant PMEs are highly relevant for plant biologists as well as for food technologists and are intensively studied in both fields. This review paper provides a structured, comprehensive overview of the knowledge accumulated over the years with regard to PME and PMEI. Attention is paid to both well-established and novel data concerning (i) their occurrence, polymorphism and physicochemical properties, (ii) primary and three-dimensional protein structures, (iii) catalytic and inhibitory activities, (iv) physiological roles in vivo and (v) relevance of (endogenous and exogenous) enzyme and inhibitor in the (food) industry. Remaining research challenges are indicated.     

Regulation and role in pathogenicity of Erwinia chrysanthemi 3937 pectin methylesterase.

The gene pem, encoding the pectin methylesterase (PME) of Erwinia chrysanthemi 3937, was cloned and mutagenized by mini-Mu transposable elements. A second gene, pecY, which could act as a negative regulator of PME was found 5' to the pem gene. A PME-E. chrysanthemi derivative inoculate onto Saintpaulia plants was shown to be clearly noninvasive, demonstrating the important role of this enzyme in soft rot disease.     

Purification, characterization, thermal, and high-pressure inactivation of pectin methylesterase from bananas (cv Cavendish)

Pectin methylesterase (PME) was extracted from bananas (cv Cavendish) and purified by affinity chromatography on a CNBr-Sepharose-PME inhibitor (PMEI) column. A single protein and PME activity peak was obtained. For banana PME, a biochemical characterization in terms of molar mass (MM), pI, and kinetic parameters was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal inactivation of purified banana PME could be described by a first-order kinetic model in a temperature range of 65 degrees to 72.5 degrees C, whereas its isobaric-isothermal inactivation followed a fractional-conversion model. Banana PME was found to be more thermally stable compared with PMEs extracted from orange, tomato, and apple.     

Mago nashi Interacts with a Taiwania (Taiwania cryptomerioides) Pectin Methylesterase-like Protein

Mago nashi proteins are highly conserved among eukaryotes. They are involved in oogenesis, embryogenesis and germ-line determination during animal development, and play important roles in pollen tube growth, root development and spermatogenesis during plant development. In this study, we used yeast two-hybrid screening to show that the TcMago protein can interact with a Taiwania (Taiwania cryptomerioides) pectin methylesterase-like protein (TcPME1) which consists of a transmembrane domain, a pectin methylesterase inhibitor (PMEI) domain and a pectin methylesterase (PME) domain. The PME domain of TcPME1 was necessary for binding with the TcMago protein. The PME domain was highly conserved in all the plants assayed and had five well conserved active site residues. The predicted protein tertiary structures revealed that the PMEI domain and PME domain of TcPME1 are similar to kiwi (Actinidia deliciosa) PMEI and carrot (Daucus carota) PME, respectively. TcPME1 was expressed abundantly in the early stage of root elongation and accumulated at root tip. Moreover, TcPME1 expression was inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Thus, TcPME1 might be involved in root elongation, shoot development and auxin transport during Taiwania development.     

Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.     

Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB

The crystal structure of the unphosphorylated state of methylesterase CheB shows that the regulatory domain blocks access of substrate to the active site of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of approximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deuterium exchange. No changes in solvent accessibility were seen in the regulatory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two regions flank the active site and contain domain-domain contact residues. Comparison with results from the isolated catalytic domain-containing C-terminal fragment of CheB (residues 147-349) showed that the increased solvent accessibility was less than would have occurred upon detachment of the regulatory domain. Thus, phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of CheB.     

Crystal structure of human uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. This activity is essential in all organisms, and subnormal activity of URO-D leads to the most common form of porphyria in humans, porphyria cutanea tarda (PCT). We have determined the crystal structure of recombinant human URO-D at 1.60 A resolution. The 40.8 kDa protein is comprised of a single domain containing a (beta/alpha)8-barrel with a deep active site cleft formed by loops at the C-terminal ends of the barrel strands. Many conserved residues cluster at this cleft, including the invariant side chains of Arg37, Arg41 and His339, which probably function in substrate binding, and Asp86, Tyr164 and Ser219, which may function in either binding or catalysis. URO-D is a dimer in solution (Kd = 0.1 microM), and this dimer also appears to be formed in the crystal. Assembly of the dimer juxtaposes the active site clefts of the monomers, suggesting a functionally important interaction between the catalytic centers.     

Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor

Thermococcus litoralis 4-alpha-glucanotransferase (TLGT) belongs to glucoside hydrolase family 57 and catalyzes the disproportionation of amylose and the formation of large cyclic alpha-1,4-glucan (cycloamylose) from linear amylose. We determined the crystal structure of TLGT with and without an inhibitor, acarbose. TLGT is composed of two domains: an N-terminal domain (domain I), which contains a (beta/alpha)7 barrel fold, and a C-terminal domain (domain II), which has a twisted beta-sandwich fold. In the structure of TLGT complexed with acarbose, the inhibitor was bound at the cleft within domain I, indicating that domain I is a catalytic domain of TLGT. The acarbose-bound structure also clarified that Glu123 and Asp214 were the catalytic nucleophile and acid/base catalyst, respectively, and revealed the residues involved in substrate binding. It seemed that TLGT produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft. Interestingly, domain I of TLGT shares some structural features with the catalytic domain of Golgi alpha-mannosidase from Drosophila melanogaster, which belongs to glucoside hydrolase family 38. Furthermore, the catalytic residue of the two enzymes is located in the same position. These observations suggest that families 57 and 38 evolved from a common ancestor.     

Site-directed mutagenesis of the active site of pectin methylesterase from Aspergillus niger RH5344

Summary The role of two histidine residues of pectin methylesterase (PME) were analysed by site-directed mutagenesis. Mutant and wild-type pmeA-cDNA were expressed in A. niger strain NRRL3. Both mutant enzymes exhibited the same mobility on SDS-polyacrylamide gel electrophoresis and gave similar circular dichroism spectra to that of the wild-type enzyme. Substitution of His-137 to alanine caused a loss of PME activity. In contrast, replacement of His-188 had no effect on the PME activity. These results revealed that the histidine residue at position 137 is essential for enzyme activity and probably located in the active site of PME.     

Pectin methylesterase inhibitor

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.     

1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis.

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel beta-helix with 10 complete turns. The beta-helix is composed of four parallel beta-sheets, and has one very small alpha-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the beta-helix. Site-directed mutagenesis of Asp(180), Asp(201), Asp(202), His(223), Arg(256), and Lys(258), which are located in this cleft, results in a severe reduction of activity, demonstrating that these residues are important for substrate binding and/or catalysis. The juxtaposition of the catalytic residues differs from that normally encountered in inverting glycosyl hydrolases. A comparison of the endopolygalacturonase II active site with that of the P22 tailspike rhamnosidase suggests that Asp(180) and Asp(202) activate the attacking nucleophilic water molecule, while Asp(201) protonates the glycosidic oxygen of the scissile bond.     

Perfusion chromatography separation of the tomato fruit-specific pectin methylesterase from a semipurified commercial enzyme preparation

A rapid and simple method was developed, using perfusion chromatography media, to separate the fruit-specific pectin methylesterase (PME) isoform from the depolymerizing enzyme polygalacturonase (PG) and other contaminating pectinases present in a commercial tomato enzyme preparation. Pectinase activities were adsorbed onto a Poros HS (a strong cation exchanger) column in 20 M HEPES buffer at pH 7.5. The fruit-specific PME was eluted from the column with 80 mM NaCl, followed by a step to 300 mM NaCl to elute PG activity. Rechromatography of the PME activity peak with a linear gradient further resolved two PME isoenzymes and removed residual traces of PG activity. The PG activity peak was further treated with lectin affinity chromatography to provide purified PG enzyme, which was separated from a salt-dependent PME (tentatively identified as a "ubiquitous-type" isoform), and a pectin acetylesterase. The later enzyme has not been reported previously in tomato. This method provides monocomponent enzymes that will be useful for studying enzyme mechanisms and for modifying pectin structure and functional properties.     

Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies

Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 mM divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein during plant-pathogen interactions.