Tomoyuki Konishi

Molecular Cloning of a β-Galactosidase from Radish That Specifically Hydrolyzes β-(1→3)- and β-(1→6)-Galactosyl Residues of Arabinogalactan Protein

A basic β-galactosidase with high specificity toward β-(1→3)- and β-(1→6)-galactosyl residues was cloned from radish (Raphanus sativus) plants by reverse transcription-PCR. The gene, designated RsBGAL1, contained an open reading frame consisting of 2,532 bp (851 amino acids). It is expressed in hypocotyls and young leaves. RsBGAL1 was highly similar to β-galactosidases having exo-β-(1→4)-galactanase activity found in higher plants and belongs to family 35 of the glycosyl hydrolases. Recombinant RsBGAL1 was expressed in Pichia pastoris and purified to homogeneity. The recombinant enzyme specifically hydrolyzed β-(1→3)- and β-(1→6)-galactooligosaccharides, the same substrates as the native enzyme isolated from radish seeds (Sekimata et al., 1989). It split off about 90% of the carbohydrate moieties of an arabinogalactan protein extracted from radish roots in concerted action with microbial α-l-arabinofuranosidase and β-glucuronidase. These results suggest that RsBGAL1 is a new kind of β-galactosidase with different substrate specificity than other β-galactosidases that exhibit exo-β-(1→4)-galactanase activity. The C-terminal region (9.6 kD) of RsBGAL1 is significantly similar to the Gal lectin-like domain, but this region is not retained in the native enzyme. Assuming posttranslational processing of RsBGAL1 with elimination of the Gal lectin-like domain results in a protein consisting of two subunits with molecular masses of 46 and 34 kD (calculated from the RsBGAL1 gene sequence). This is in good agreement with the SDS-PAGE and matrix-assisted laser desorption/ionization-time-of flight mass spectrometry measurements for subunits of the native enzyme (45 and 34 kD) and may thus partially explain the formation process of the native enzyme.

Properties and physiological functions of UDP-sugar pyrophosphorylase in Arabidopsis.

UDP-sugar pyrophosphorylase catalyzes the conversion of various monosaccharide 1-phosphates to the respective UDP-sugars in the salvage pathway. Using the genomic database, we cloned a putative gene for UDP-sugar pyrophosphorylase from Arabidopsis. Although relatively stronger expression was detected in the vascular tissue of leaves and the pollen, AtUSP is expressed in most cell types of Arabidopsis, indicating a housekeeping function in nucleotide sugar metabolism. Recombinant AtUSP expressed in Escherichia coli exhibited broad specificity toward monosaccharide 1-phosphates, resulting in the formation of various UDP-sugars such as UDP-glucose, -galactose, -glucuronic acid, -xylose and -L-arabinose. A loss-of-function mutation in the AtUSP gene caused by T-DNA insertion completely abolished male fertility. These results indicate that AtUSP functions as a UDP-sugar pyrophosphorylase in the salvage pathway, and that the generation of UDP-sugars from monosaccharide 1-phosphates catalyzed by AtUSP is essential for pollen development in Arabidopsis.

Defects in flagellin glycosylation affect the virulence of Pseudomonas syringae pv. tabaci 6605.

Flagellar motility and its glycosylation are indispensable for the virulence of Pseudomonas syringae pv. tabaci 6605. Six serine residues of the flagellin protein at positions 143, 164, 176, 183, 193 and 201 are glycosylated, and the glycan structure at 201 was determined to consist of a trisaccharide of two L-rhamnosyl residues and a modified 4-amino-4,6-dideoxyglucosyl (viosamine) residue. To investigate the glycan structures attached to the other serine residues and to identify the glycans important for virulence, Ser/Ala-substituted mutants were generated. Six mutant strains that each retained a single glycosylated serine residue were generated by replacing five of the six serine residues with alanine residues. MALDI-TOF mass analysis of flagellin proteins revealed that the major component of each glycan was a trisaccharide basically similar to that at position 201, but with heterogeneity in glycoform distribution. Swarming motility and amounts of acylhomoserine lactones (AHLs) as quorum-sensing signal molecules were significantly reduced, especially in the S143-5S/A, S164-5S/A and S201-5S/A mutants, whereas tolerance to antibiotics was increased in these three mutants. All the mutants showed lower ability to cause disease on host tobacco plants. These results supported our previous finding that glycosylation of the most externally located sites on the surface of the flagellin molecule, such as S176 and S183, is required for virulence in P. syringae pv. tabaci 6605. Furthermore, it is speculated that flagellum-dependent motility might be correlated with quorum sensing and antibiotic resistance.

Properties of family 79 β-glucuronidases that hydrolyze β-glucuronosyl and 4-O-methyl-β-glucuronosyl residues of arabinogalactan-protein

The carbohydrate moieties of arabinogalactan-proteins (AGPs), which are mainly composed of Gal, L-Ara, GlcA, and 4-Me-GlcA residues, are essential for the physiological functions of these proteoglycans in higher plants. For this study, we have identified two genes encoding family 79 beta-glucuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native beta-glucuronidase purified from a commercial pectolytic enzyme preparation from A. niger. Although the deduced protein sequences of AnGlcAase and NcGlcAase were highly similar, the recombinant enzymes expressed in Pichia pastoris exhibited distinct substrate specificity toward 4-Me-GlcA residues of AGPs: recombinant AnGlcAase (rAnGlcAase) substantially liberated both GlcA and 4-Me-GlcA residues from radish AGPs, whereas recombinant NcGlcAase (rNcGlcAase) activity on the 4-Me-GlcA residues of AGPs was very low. Maximum activity of rAnGlcAase hydrolyzing PNP beta-GlcA occurred at pH 3.0-4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent Km values of rAnGlcAase were 30.4 microM for PNP beta-GlcA and 422 microM for beta-GlcA-(1-->6)-Gal, and those of rNcGlcAase were 38.3 microM and 378 microM, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP beta-GlcA to various monosaccharide acceptors such as Glc, Gal, and Xyl. We propose that both AnGlcAase and NcGlcAase are instances of a novel type of beta-glucuronidase with the capacity to hydrolyze beta-GlcA and 4-Me-beta-GlcA residues of AGPs, although they differ significantly in their preferences.

An arginyl residue in rice UDP-arabinopyranose mutase is required for catalytic activity and autoglycosylation

Plants use UDP-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing complex carbohydrates. UDP-Araf itself is formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). However, the mechanism by which this enzyme catalyzes the interconversion of UDP-Arap and UDP-Araf has not been determined. To gain insight into this reaction, functionally recombinant rUAMs were reacted with UDP-Glc or UDP-Araf. The glycosylated recombinant UAMs were fragmented with trypsin, and the glycopeptides formed were then identified and sequenced by LC-MS/MS. The results of these experiments, together with site-directed mutagenesis studies, suggest that in functional UAMs an arginyl residue is reversibly glycosylated with a single glycosyl residue, and that this residue is required for mutase activity. We also provide evidence that a DXD motif is required for catalytic activity.

Down-regulation of UDP-arabinopyranose mutase reduces the proportion of arabinofuranose present in rice cell walls.

Arabinoxylans may account for up to 25% of the mass of grass cell walls. The interactions of these polysaccharides with themselves and with cellulose and lignin is believed to affect the walls physical properties and increase the walls resistance to biochemical conversion to fermentable sugars. Arabinoxylans have a backbone composed of 1,4-linked β-D-xylosyl residues, some of which are substituted at O-2 or O-3 with single arabinofuranosyl (Araf) residues. The Araf residues are likely transferred from UDP-Araf to the xylan backbone by arabinofuranosyltransferases. UDP-Araf is itself formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). In this study, RNA interference (RNAi) was used to suppress UAM expression in rice plants and thereby reduce the amounts of UDP-Araf available for cell wall synthesis. Several of the transgenic plants had reduced proportions of Araf in their walls together with a decrease in the extent of substitution of the xylan backbone, and a reduction of between 25% and 80% in ferulic acid and p-coumaric acid contents of the cell walls. Those transgenic plants with >25% reduction in the amounts of Araf were dwarfed and infertile.

Purification and Biochemical Characterization of Recombinant Rice UDP-Arabinopyranose Mutase Generated in Insect Cells

Plants utilize UDP-arabinofuranose (UDP-Araf) in the biosynthesis of Araf-containing complex carbohydrates. UDP-Araf is synthesized from UDP-arabinopyranose by UDP-arabinopyranose mutases (UAMs). Here we describe the heterologous expression of rice (Oryza sativa) UAM genes in insect cells and report some of their enzymatic properties. Recombinant UAMs might serve as useful tools for the biosynthesis of UDP-Araf and might be better than chemical synthesis.

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin.

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rha-(1-->, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rha-(1-->, which was identified in a previous study.