Rodjana Opassiri

Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 β-glucosidase

BackgroundGlycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice.ResultsForty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3–6 glucose residues and laminaribiose.ConclusionCareful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.

Structural Insights into Rice BGlu1 β-Glucosidase Oligosaccharide Hydrolysis and Transglycosylation

The structures of rice BGlu1 beta-glucosidase, a plant beta-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 A and 1.55 A resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) beta-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long beta-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa beta-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-beta-glucanase/beta-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/beta-II beta-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.

Characterization of a rice β-glucosidase highly expressed in flower and germinating shoot

The cDNAs for two β-glucosidase (E.C. 3.2.1.21) isozymes from rice (Oryza sativa L.), designated bglu1 and bglu2, were cloned and sequenced. The cDNA sequences for bglu1 and bglu2 included open reading frames encoding 504 and 500 amino acid precursor proteins, respectively. Both of these enzymes appeared to enter the secretory pathway, as judged by their N-terminal signal sequences. Southern blots using gene-specific probes indicated that bglu1 and bglu2 were single copy genes. The bglu1 and bglu2 mRNAs were highly expressed in the shoot during germination, with a similar time course. However, differences were seen in expression in mature plants, where bglu1 was highly expressed in flowers, but bglu2 was not. A recombinant thioredoxin fusion protein produced from the bglu1 cDNA in redox-deficient Escherichia coli (BGlu1) hydrolyzed p-nitrophenol β-d-glucoside, β-d-fucoside, and other p-nitrophenol β-d-glycosides, and was strongly inhibited by glucono-1,5-lactone. It also hydrolyzed some natural glucosides, including the rice-derived pyridoxine-5′-O-β-d-glucoside, and hydrolyzed and transglycosylated short β-(1→3) and β-(1→4) linked gluco-oligosaccharides. Based on the results, possible functions of BGlu1 include hydrolysis and recycling of oligosaccharides generated from rapid cell wall expansion during seed germination and flower expansion, and release of the coenzyme pyridoxine from its glucose-conjugated storage form.

Structural and Enzymatic Characterization of Os3BGlu6, a Rice β-Glucosidase Hydrolyzing Hydrophobic Glycosides and (1→3)- and (1→2)-Linked Disaccharides

Glycoside hydrolase family 1 (GH1) β-glucosidases play roles in many processes in plants, such as chemical defense, alkaloid metabolism, hydrolysis of cell wall-derived oligosaccharides, phytohormone regulation, and lignification. However, the functions of most of the 34 GH1 gene products in rice (Oryza sativa) are unknown. Os3BGlu6, a rice β-glucosidase representing a previously uncharacterized phylogenetic cluster of GH1, was produced in recombinant Escherichia coli. Os3BGlu6 hydrolyzed p-nitrophenyl (pNP)-β-d-fucoside (kcat/Km = 67 mm−1 s−1), pNP-β-d-glucoside (kcat/Km = 6.2 mm−1 s−1), and pNP-β-d-galactoside (kcat/Km = 1.6 mm−1s−1) efficiently but had little activity toward other pNP glycosides. It also had high activity toward n-octyl-β-d-glucoside and β-(1→3)- and β-(1→2)-linked disaccharides and was able to hydrolyze apigenin β-glucoside and several other natural glycosides. Crystal structures of Os3BGlu6 and its complexes with a covalent intermediate, 2-deoxy-2-fluoroglucoside, and a nonhydrolyzable substrate analog, n-octyl-β-d-thioglucopyranoside, were solved at 1.83, 1.81, and 1.80 Å resolution, respectively. The position of the covalently trapped 2-F-glucosyl residue in the enzyme was similar to that in a 2-F-glucosyl intermediate complex of Os3BGlu7 (rice BGlu1). The side chain of methionine-251 in the mouth of the active site appeared to block the binding of extended β-(1→4)-linked oligosaccharides and interact with the hydrophobic aglycone of n-octyl-β-d-thioglucopyranoside. This correlates with the preference of Os3BGlu6 for short oligosaccharides and hydrophobic glycosides.

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Background: Glycosylation regulates the activities of plant metabolites and is mediated by glycosyltransferases (GT), glycoside hydrolases (GH), and transglycosidases (TG). Results: The vacuolar TG Os9BGlu31 transfers glucose between phenolic acid esters and other compounds. Conclusion: Os9BGlu31 equilibrates phenolic acids, phytohormones, and their glucosyl conjugates. Significance: Os9BGlu31 and similar TG can broaden glycoconjugate diversity in planta. Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu169), catalytic nucleophile residues (Glu387), and His386, the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

A stress-induced rice (Oryza sativa L.) β-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain

GH5BG, the cDNA for a stress-induced GH5 (glycosyl hydrolase family 5) beta-glucosidase, was cloned from rice (Oryza sativa L.) seedlings. The GH5BG cDNA encodes a 510-amino-acid precursor protein that comprises 19 amino acids of prepeptide and 491 amino acids of mature protein. The protein was predicted to be extracellular. The mature protein is a member of a plant-specific subgroup of the GH5 exoglucanase subfamily that contains two major domains, a beta-1,3-exoglucanase-like domain and a fascin-like domain that is not commonly found in plant enzymes. The GH5BG mRNA is highly expressed in the shoot during germination and in leaf sheaths of mature plants. The GH5BG was up-regulated in response to salt stress, submergence stress, methyl jasmonate and abscisic acid in rice seedlings. A GUS (glucuronidase) reporter tagged at the C-terminus of GH5BG was found to be secreted to the apoplast when expressed in onion (Allium cepa) cells. A thioredoxin fusion protein produced from the GH5BG cDNA in Escherichia coli hydrolysed various pNP (p-nitrophenyl) glycosides, including beta-D-glucoside, alpha-L-arabinoside, beta-D-fucoside, beta-D-galactoside, beta-D-xyloside and beta-D-cellobioside, as well as beta-(1,4)-linked glucose oligosaccharides and beta-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/K(m)) for hydrolysis of beta-(1,4)-linked oligosaccharides by the enzyme remained constant as the DP (degree of polymerization) increased from 3 to 5. This substrate specificity is significantly different from fungal GH5 exoglucanases, such as the exo-beta-(1,3)-glucanase of the yeast Candida albicans, which may correlate with a marked reduction in a loop that makes up the active-site wall in the Candida enzyme.

Rice family GH1 glycoside hydrolases with β-D-glucosidase and β-D-mannosidase activities

Plant beta-D-mannosidases and a rice beta-D-glucosidase, Os3BGlu7, with weak beta-D-mannosidase activity, cluster together in phylogenetic analysis. To investigate the relationship between substrate specificity and amino acid sequence similarity in family GH1 glycoside hydrolases, Os3BGlu8 and Os7BGlu26, putative rice beta-D-glucosidases from this cluster, and a beta-D-mannosidase from barley (rHvBII), were expressed in Escherichia coli and characterized. Os3BGlu8, the amino acid sequence and molecular model of which are most similar to Os3BGlu7, hydrolysed 4-nitrophenyl-beta-D-glucopyranoside (4NPGlc) faster than 4-nitrophenyl-beta-D-mannopyranoside (4NPMan), while Os7BGlu26, which is most similar to rHvBII by these criteria, hydrolysed 4NPMan faster than 4NPGlc. All the enzymes hydrolyzed cellooligosaccharides with increased hydrolytic rates as the degree of polymerization increased from 3-6, but only rHvBII hydrolyzed cellobiose with a higher k(cat)/K(m) value than cellotriose. This was primarily due to strong binding of glucosyl residues at the+2 subsite for the rice enzymes, and unfavorable interactions at this subsite with rHvBII.

Expression of an endo-(1,3;1,4)-β-glucanase in response to wounding, methyl jasmonate, abscisic acid and ethephon in rice seedlings.

We isolated two rice endo-(1,3;1,4)-beta-glucanase genes, denoted OsEGL1 and OsEGL2, which encoded proteins that shared 64% amino acid sequence identity. Both the OsEGL1 and OsEGL2 genes were successfully expressed in Escherichia coli to produce functional proteins. Purified OsEGL1 and OsEGL2 proteins hydrolyzed (1,3;1,4)-beta-glucans, but not (1,3;1,6)-beta-linked or (1,3)-beta-linked glucopolysaccharides nor carboxymethyl cellulose, similar to previously characterized grass endo-(1,3;1,4)-beta-glucanases. RNA blot analysis revealed that the OsEGL1 gene is expressed constitutively not only in young roots of rice seedlings, but also in mature roots of adult rice plants. Little or no expression of the OsEGL2 gene was observed in all tissues or treatments tested, but database and RT-PCR analysis indicated it is expressed in ripening panicle. In rice seedling leaves, OsEGL1 gene expression significantly increased in response to methyl jasmonate, abscisic acid, ethephon and mechanical wounding. Mechanical wounding also increased the leaf elongation rate in rice seedlings by 16% relative to that of control seedlings at day 4 after treatment. The increase in the leaf elongation rate of rice seedlings treated under mechanical wounding was concomitant with an increase in OsEGL1 expression levels in seedling leaves.

The crystal structure of rice (Oryza sativa L.) Os4BGlu12, an oligosaccharide and tuberonic acid glucoside-hydrolyzing β-glucosidase with significant thioglucohydrolase activity

Rice Os4BGlu12, a glycoside hydrolase family 1 (GH1) β-glucosidase, hydrolyzes β-(1,4)-linked oligosaccharides of 3-6 glucosyl residues and the β-(1,3)-linked disaccharide laminaribiose, as well as certain glycosides. The crystal structures of apo Os4BGlu12, and its complexes with 2,4-dinitrophenyl-2-deoxyl-2-fluoroglucoside (DNP2FG) and 2-deoxy-2-fluoroglucose (G2F) were solved at 2.50, 2.45 and 2.40Å resolution, respectively. The overall structure of rice Os4BGlu12 is typical of GH1 enzymes, but it contains an extra disulfide bridge in the loop B region. The glucose ring of the G2F in the covalent intermediate was found in a (4)C(1) chair conformation, while that of the noncovalently bound DNP2FG had a (1)S(3) skew boat, consistent with hydrolysis via a (4)H(3) half-chair transition state. The position of the catalytic nucleophile (Glu393) in the G2F structure was more similar to that of the Sinapsis alba myrosinase G2F complex than to that in covalent intermediates of other O-glucosidases, such as rice Os3BGlu6 and Os3BGlu7 β-glucosidases. This correlated with a significant thioglucosidase activity for Os4BGlu12, although with 200- to 1200-fold lower k(cat)/K(m) values for S-glucosides than the comparable O-glucosides, while hydrolysis of S-glucosides was undetectable for Os3BGlu6 and Os3BGlu7.

Expression, purification, crystallization and preliminary X‐ray analysis of rice (Oryza sativa L.) Os4BGlu12 β‐glucosidase

Rice (Oryza sativa L.) Os4BGlu12, a glycoside hydrolase family 1 beta-glucosidase (EC 3.2.1.21), was expressed as a fusion protein with an N-terminal thioredoxin/His(6) tag in Escherichia coli strain Origami B (DE3) and purified with subsequent removal of the N-terminal tag. Native Os4BGlu12 and its complex with 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-glucopyranoside (DNP2FG) were crystallized using 19% polyethylene glycol (3350 or 2000, respectively) in 0.1 M Tris-HCl pH 8.5, 0.16 M NaCl at 288 K. Diffraction data sets for the apo and inhibitor-bound forms were collected to 2.50 and 2.45 A resolution, respectively. The space group and the unit-cell parameters of the crystal indicated the presence of two molecules per asymmetric unit, with a solvent content of 50%. The structure of Os4BGlu12 was successfully solved in space group P4(3)2(1)2 by molecular replacement using the white clover cyanogenic beta-glucosidase structure (PDB code 1cbg) as a search model.