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

BACKGROUND: Glycosyl 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. RESULTS: Forty 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. CONCLUSION: Careful 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

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

Recombinant rice Os4BGlu12 β-glucosidase purified from E. coli was crystallized with and without 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-d-glucopyranoside

รายงานการวิจัยการตรวจหาด้วยเอนไซม์และการศึกษาสารประเภทไกลโคไซด์จากพืชไทย

Suranaree University of Technolog

รายงานการวิจัยการศึกษาคุณลักษณะและการแสดงออกของเอนไซม์กลุ่มไกลโคซิลไฮโดรเลสจากพืชไทย

Suranaree University of Technolog

Rice family GH1 glycoside hydrolases with beta-d-glucosidase and beta-d-mannosidase activities

Copyright © 2009 Elsevier Inc. All rights reserved.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.Teerachai Kuntothom, Sukanya Luang, Andrew J. Harvey, Geoffrey B. Fincher, Rodjana Opassiri, Maria Hrmova, James R. Ketudat Cairn

Purification, crystallization and preliminary X-ray analysis of rice BGlu1 β-glucosidase with and without 2-deoxy-2-fluoro-β-d-glucoside

Rice BGlu1 β-glucosidase was purified from recombinant E. coli and crystallized with and without the inhibitor 2-deoxy-2-fluoro-β-d-glucose. The crystals diffracted to 2.15 and 2.75 Å, respectively

Beta-glucosidase, exo-beta-glucanase and pyridoxine transglucosylase activities of rice BGlu1.

The bglu1 cDNA for a beta-glucosidase cloned from rice (Oryza sativa L.) seedlings was expressed as a soluble and active protein in Escherichia coli and designated BGlu1. This enzyme hydrolysed beta-1,4-linked oligosaccharides with increasing catalytic efficiency (kcat/Km) values as the DP (degree of polymerization) increased from 2 to 6. In contrast, hydrolysis of beta-1,3-linked oligosaccharides decreased from DP 2 to 3, and polymers with a DP greater than 3 were not hydrolysed. The enzyme also hydrolysed p -nitrophenyl beta-D-glycosides and some natural glucosides but with lower catalytic efficiency than beta-linked oligosaccharides. Pyridoxine 5'-O-beta-D-glucoside was the most efficiently hydrolysed natural glycoside tested. BGlu1 also had high transglucosylation activity towards pyridoxine, producing pyridoxine 5'-O-beta-D-glucopyranoside in the presence of the glucose donor p-nitrophenyl beta-D-glucoside

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) β-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 β-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 β-D-glucoside, α-L-arabinoside, β-D-fucoside, β-D-galactoside, β-D-xyloside and β-D-cellobioside, as well as β-(1,4)-linked glucose oligosaccharides and β-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/Km) for hydrolysis of β-(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-β-(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