Substrate-recognition mechanism of tomato ß-galactosidase 4 using X-ray crystallography and docking simulation.

MAIN CONCLUSION: TBG4 recognize multiple linkage types substrates due to having a spatially wide subsite + 1. This feature allows the degradation of AGI, AGII, and AGP leading to the fruit ripening. ß-galactosidase (EC 3. 2. 1. 23) catalyzes the hydrolysis of ß-galactan and release of D-galactose. Tomato has at least 17 ß-galactosidases (TBGs), of which, TBG 4 is responsible for fruit ripening. TBG4 hydrolyzes not only ß-1,4-bound galactans, but also ß-1,3- and ß-1,6-galactans. In this study, we compared each enzyme-substrate complex using X-ray crystallography, ensemble refinement, and docking simulation to understand the broad substrate-specificity of TBG4. In subsite - 1, most interactions were conserved across each linkage type of galactobioses; however, some differences were seen in subsite + 1, owing to the huge volume of catalytic pocket. In addition to this, docking simulation indicated TBG4 to possibly have more positive subsites to recognize and hydrolyze longer galactans. Taken together, our results indicated that during tomato fruit ripening, TBG4 plays an important role by degrading arabinogalactan I (AGI), arabinogalactan II (AGII), and the carbohydrate moiety of arabinogalactan protein (AGP).

Characterization of three GH35 ß-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B.

Three recombinant ß-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze ß-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-ß-D-galactopyranoside and ß-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the ß-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked ß-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Structural and functional analysis of tomato ß-galactosidase 4: insight into the substrate specificity of the fruit softening-related enzyme.

Plant ß-galactosidases hydrolyze cell wall ß-(1,4)-galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato ß-galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of ß-(1,4)-galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1-7 that belongs to the ß-galactosidase/exo-ß-(1,4)-galactanase subfamily. The enzyme can hydrolyze a wide range of plant-derived (1,4)- or 4-linked polysaccharides, and shows a strong ability to attack ß-(1,4)-galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with ß-d-galactose. TBG4 comprises a catalytic TIM barrel domain followed by three ß-sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 ß-galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards ß-(1,6)-galactobiose, and ~0.6-fold activity towards ß-(1,4)-galactobiose, compared with wild-type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant ß-galactosidases towards ß-1,4 and ß-1,6 linkages.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of tomato ß-galactosidase 4.

Plant ß-galactosidases play important roles in carbohydrate-reserve mobilization, cell-wall expansion and degradation, and turnover of signalling molecules during ripening. Tomato ß-galactosidase 4 (TBG4) not only has ß-galactosidase activity but also has exo-ß-(1,4)-galactanase activity, and prefers ß-(1,4)-galactans longer than pentamers as its substrates; most other ß-galactosidases only have the former activity. Recombinant TBG4 protein expressed in the yeast Pichia pastoris was crystallized by the sitting-drop vapour-diffusion method using PEG 10,000 as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-parameters a = 92.82, b = 96.30, c = 159.26 Å, and diffracted to 1.65 Å resolution. Calculation of the Matthews coefficient suggested the presence of two monomers per asymmetric unit (VM = 2.2 Å(3) Da(-1)), with a solvent content of 45%.

Enzymatic activity and substrate specificity of the recombinant tomato ß-galactosidase 1.

The open reading frame of tomato ß-galactosidase 1 was expressed in yeast, and the enzymatic properties and substrate specificity were investigated. The enzyme had peak activity at pH 5.0 and 40-50°C. TBG1 was active on ß-(1,3)- and ß-(1,6)-galactobiose and lactose. TBG1 released galactose from lupin galactan, tomato fruit alkali soluble pectin, arabinogalactan, gum arabic and methyl ß-(1,6)-galactohexaoside, but not from labeled ß-(1,4)-galactoheptaose. TBG1 was assessed for its ability to degrade three galactosyl-containing cell wall fractions purified from different development and ripening stages of tomato fruit. TBG1 released galactose from all of the fractions from all of the stages tested. TBG1 activity was highest on the hemicellulose fraction at the 10 and 20d after pollination stage. This result is not correlated the with TBG1 expression pattern. TBG1 might act on a small but specific set of polysaccharide containing galactose.

Peculiarities and applications of galactanolytic enzymes that act on type I and II arabinogalactans.

Arabinogalactans (AGs) are branched galactans to which arabinose residues are bound as side chains and are widely distributed in plant cell walls. They can be grouped into two types based on the structures of their backbones. Type I AGs have ß-1,4-galactan backbones and are often covalently linked to the rhamnogalacturonan-I region of pectins. Type II AGs have ß-1,3-galactan backbones and are often covalently linked to proteins. The main enzymes involved in the degradation of AGs are endo-ß-galactanases, exo-ß-galactanases, and ß-galactosidases, although other enzymes such as α-L-arabinofuranosidases, ß-L-arabinopyranosidases, and ß-D-glucuronidases are required to remove the side chains for efficient degradation of the polysaccharides. Galactanolytic enzymes have a wide variety of potential uses, including the bioconversion of AGs to fermentable sugars for production of commodity chemicals like ethanol, biobleaching of cellulose pulp, modulation of pectin properties, improving animal feed, and determining the chemical structure of AGs. This review summarizes our current knowledge about the biochemical properties and potential applications of AG-degrading enzymes.

Characterization of an exo-ß-1,3-D: -galactanase from Sphingomonas sp. 24T and its application to structural analysis of larch wood arabinogalactan.

A type II arabinogalactan-degrading enzyme, termed Exo-1,3-Gal, was purified to homogeneity from the culture filtrate of Sphingomonas sp. 24T. It has an apparent molecular mass of 48 kDa by SDS-PAGE. Exo-1,3-Gal was stable from pH 3 to 10 and at temperatures up to 40 °C. The optimum pH and temperature for enzyme activity were pH 6 to 7 and 50 °C, respectively. Galactose was released from ß-1,3-D: -galactan and ß-1,3-D: -galactooligosaccharides by the action of Exo-1,3-Gal, indicating that the enzyme was an exo-ß-1,3-D: -galactanase. Analysis of the reaction products of ß-1,3-galactotriose by high-performance anion-exchange chromatography revealed that the enzyme hydrolyzed the substrate in a non-processive mode. Exo-1,3-Gal bypassed the branching points of ß-1,3-galactan backbones in larch wood arabinogalactan (LWAG) to produce mainly galactose, ß-1,6-galactobiose, and unidentified oligosaccharides 1 and 2 with the molar ratios of 7:19:62:12. Oligosaccharides 1 and 2 were enzymatically determined to be ß-1,6-galactotriose and ß-1,6-galactotriose substituted with a single arabinofuranose residue, respectively. The ratio of side chains enzymatically released from LWAG was in good agreement with the postulated structure of the polysaccharide previously determined by chemical methods.

Synthesis and structure of dinuclear hafnium(IV) and zirconium(IV) complexes sandwiched between 2 mono-lacunary alpha-Keggin polyoxometalates.

The synthesis and characterization of two dinuclear HfIV and ZrIV complexes sandwiched between 2 mono-lacunary alpha-Keggin polyoxometalates (POMs), i.e., (Et2NH2)8[{alpha-PW11O39Hf(micro-OH)(H2O)}2].7H2O (Et2NH(2)-1) and (Et2NH2)8[{alpha-PW11O39Zr(micro-OH)(H2O)}2].7H2O (Et2NH(2)-2), are described. [Note: the moieties of their polyoxoanions are abbreviated simply as and , respectively.] A pair of HfIV- and ZrIV-containing POMs belonging to the same family were herein isolated as diethylammonium salts and were unambiguously characterized by complete elemental analysis, including sodium and oxygen analyses, TG/DTA, FT-IR, single-crystal X-ray structure analysis and solution (31P and 183W) NMR spectroscopy. Polyoxoanions 1 and 2 were isostructural with each other. The central [M2(micro-OH)2(H2O)2]6+ (M=Hf, Zr) cation unit was composed of 2 edge-sharing polyhedral M units, which were linked through 2 micro-OH groups and contained 1 water molecule coordinated to each metal center. Since the mono-lacunary Keggin POM acts as an oxygen-donor quadridentate ligand, the Hf and Zr centers are 7-coordinate. It should be noted that the present 2 Keggin 2:2-type compounds, Et2NH(2)-1 and Et2NH(2)-2, undergo a reversible conversion to Keggin 1:2-type complexes [M(alpha-PW11O39)2]10-, respectively, in solution under appropriate conditions. The synthesis of Et2NH(2)-1 and Et2NH(2)-2 is based on such an interconversion. The Zr compound Et2NH(2)-2 was rigorously compared with the 3 Zr POMs (OK-1-OK-3), recently reported by Kholdeeva's group: their POMs in a different protonation-state did not contain any coordinating water molecules.

Enzymatic activity and substrate specificity of recombinant tomato beta-galactosidases 4 and 5.

The open reading frames of tomato beta-galactosidase (TBG) 4 and 5 cDNAs were expressed in yeast, and the enzymes properties and substrate specificities were investigated. The two enzymes had peak activities between pH 4-4.5 and 37-45 degrees C. TBG4 specifically hydrolyzed beta-(1-->4) and 4-linked galactooligosaccharides. TBG5 had a strong preference to hydrolyze beta-(1-->3) and beta-(1-->6)-linked galactooligosaccharides. Exo-beta-galactanase activity of the TBG enzymes was measured by determining the release of galactosyl residues from native tomato cell wall fractions throughout fruit development and ripening. Both TBGs released galactose from all of the fractions and stages tested. TBG4 activity was highest using chelator soluble pectin and alkali soluble pectin at the turning stage of ripening. Using aminopyrene trisulfonate labeled substrates, TBG4 was the only enzyme with strong exo-beta-(1-->4)-galactanase activity on 5 mer or greater galactans. TBG4 and TBG5 were both able to degrade galactosylated rhamnogalacturonan. Neither enzyme was able to degrade galactosylated xyloglucan.

Expression of three expansin genes during development and maturation of Kyoho grape berries.

Expansins are cell-wall-localized proteins that induce loosening of isolated plant cell walls in vitro in a pH-dependent manner, but exhibit no detectable hydrolase or transglycosylase activity. Three putative expansin cDNAs, Vlexp1, Vlexp2, and Vlexp3 were isolated from a cDNA library made from mature berries of the Kyoho grape. Expression profiles of the 3 genes were analyzed throughout berry development. Accumulation of the Vlexp3 transcript was closely correlated with berry softening, and expression of this gene was detected before véraison and markedly increased at véraison (onset of berry softening). Expression of Vlexp3 was berry-specific. Vlexp1 and Vlexp2 mRNA accumulation began during the expansion stage of berry development and expression increased for both genes during ripening. Vlexp1 and Vlexp2 mRNA was detected in leaf, tendril and flower tissues and Vlexp2 mRNA was additionally detected in root and seed tissues. These findings suggest that the three expansin genes are associated with cell division or expansion and berry ripening. Vlexp3, in particular, is most likely to play a role in grape berry softening at véraison.

Inhibition of acetate ester biosynthesis in banana (Musa sapientum L.) fruit pulp under anaerobic conditions.

The effect of anaerobic conditions on acetate ester biosynthesis in ripened banana pulp was investigated. Incubation of the pulp in less than 1% O(2) resulted in a significant reduction in the formation of ethyl acetate. Regardless of the presence of a large amount of endogenous ethanol and the remaining exogenous isobutyl alcohol after complete anaerobic incubation with the pulp, the production of acetate ester decreased. The effect of addition of pyruvate, isobutyl alcohol, acetate, and methyl hexanoate on acetate ester formation in 100% N(2) was also investigated. The addition of pyruvate and isobutyl alcohol to the pulp gave lower acetate esters in N(2) than in air, whereas the pulp incubated with acetate and isobutyl alcohol produced more acetate ester in both conditions. Therefore, the lack of acetyl CoA, or more precisely acetate, in the tissue is the main reason for the inhibition of acetate ester formation under anaerobic conditions. The activity of beta-oxidation measured by incubation with methyl hexanoate was detected only in the samples incubated in air. The formation of acetyl CoA, derived from pyruvate through mitochondria and through beta-oxidation, was inhibited by anaerobic conditions, which suggests that mitochondrial activity and/or beta-oxidation are essential for ester biosynthesis.