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.

Incomplete vitamin K-dependent γ-carboxylation has been found in bone Gla protein (BGP) isolated from each of 20 different human bone samples. Using N-terminal protein sequencing of the methyl-esterified protein (Anal Biochem 1991;199:93-97), a method that directly measures the percentage of γ-carboxylation at each target glutamate residue, the extent of incomplete BGP γ-carboxylation was found to depend strongly on sequence position, with (x̄ + SD) 67 + 14% γ-carboxylation at residue 17, 88 + 9% γ-carboxylation at residue 21, and 93 + 4% γ-carboxylation at residue 24. There is a strong correlation between the incomplete γ-carboxylation at glutamate residues 17 and 21 for BGP purified from the 20 bone samples (p < 0.001), which suggests that individual differences in the efficiency of BGP γ-carboxylation during synthesis probably cause the observed differences in percentage BGP γ-carboxylation between bone samples. These results have been interpreted using a kinetic treatment of γ-carboxylation. This treatment predicts the existence of differences in the extent of γ-carboxylation between glutamate residues in BGP, as well as the correlation between percentage carboxylation at Glu¹⁷ and Glu²¹. Although the molecular basis of incomplete BGP γ-carboxylation is at present unknown, if incomplete BGP γ-carboxylation were caused only by differences in the availability of vitamin K in bone cells, this kinetic treatment predicts that the range in BGP γ-carboxylation observed in the 20 human bone samples studied here could be explained by a relatively modest fivefold range in the vitamin K levels of these individuals.

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 (k_cat/K_m = 67 mm⁻¹ s⁻¹), pNP-β-d-glucoside (k_cat/K_m = 6.2 mm⁻¹ s⁻¹), and pNP-β-d-galactoside (k_cat/K_m = 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.