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).

Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-ß-1,3-galactanase from Phanerochaete chrysosporium.

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-ß-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the ß-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes ß-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization.

Mutation of cysteine residues increases heterologous expression of peach expansin in the methylotrophic yeast Pichia pastoris.

The study of Carbohydrate-Active enZymes (CAZymes) associated with plant cell wall metabolism is important for elucidating the developmental mechanisms of plants and also for the utilization of plants as a biomass resource. The use of recombinant proteins is common in this context, but heterologous expression of plant proteins is particularly difficult, in part because the presence of many cysteine residues promotes denaturation, aggregation and/or protein misfolding. In this study, we evaluated two phenotypes of methylotrophic yeast Pichia pastoris as expression hosts for expansin from peach (Prunus persica (L.) Batsch, PpEXP1), which is one of the most challenging targets for heterologous expression. cDNAs encoding wild-type expansin (PpEXP1_WT) and a mutant in which all cysteine residues were replaced with serine (PpEXP1_CS) were each inserted into expression vectors, and the protein expression levels were compared. The total amount of secreted protein in PpEXP1_WT culture was approximately twice that of PpEXP1_CS. However, the amounts of recombinant expansin were 0.58 and 4.3 mg l-1, corresponding to 0.18% and 2.37% of total expressed protein, respectively. This 13-fold increase in production of the mutant in P. pastoris indicates that the replacement of cysteine residues stabilizes recombinant PpEXP1.

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.

beta-Citryl-L-glutamate is an endogenous iron chelator that occurs naturally in the developing brain.

The compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The stability constant, logbeta(pqr) for M(p)(beta-CG)(q)H(r) was calculated from pH titration data, which showed that beta-CG forms relatively strong complexes with Fe(III), Cu(II), Fe(II) and Zn(II). beta-CG was also found able to solubilize Fe more effectively from Fe(OH)(2) than from Fe(OH)(3). Therefore, we examined the effects of beta-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of beta-CG and metal ion-(beta-CG) complexes. beta-CG inhibited the Fe-dependent degradation of deoxyribose and Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that beta-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that beta-CG is an endogenous low molecular weight Fe chelator.

Lipid and fatty acid composition of mesocarp and seed of avocado fruits harvested at northern range in Japan.

The lipid and fatty acid composition of the mesocarp and seed of avocado fruit grown and harvested in Japan, which is located at the northern range of the avacado, was investigated and compared to an imported avocado purchased commercially. The potential of the avocado mesocarp as an agricultural product in Japan was also explored. Total lipids (TL) accounted for approximately 20% of the mesocarp. Further analysis showed that the neutral lipid (NL) fraction accounted for at least 95% of the TL, and almost 90% of NL was triacylglycerol. Monoenoic acids accounted for at least 65% of the total fatty acids, and oleic acid, which is regarded as an especially important functional component of avacado accounted for approximately 50% of the monounsaturated fatty acids. A comparison of the Japanese avocado cultivars and an imported avocado cultivar in the present study revealed no significant differences in the lipid and fatty acid compositions. Therefore, production of avocado fruit, which is rich in various nutritional components, is expected to be increased on a larger number of farms in Japan in the future. It is believed to be necessary to carry out further verification, such as the establishment of a cultivation technique adoptable to Japan, examination of optimal soil and land features, and cultivar selection.