Molecular and Structural Characterization of Hexameric β-d-Glucosidases in Wheat and Rye

Abstract

The wheat (Triticum aestivum) and rye (Secale cereale) b-D-glucosidases hydrolyze hydroxamic acid-glucose conjugates, exist as different types of isozyme, and function as oligomers. In this study, three cDNAs encoding b-D-glucosidases (TaGlu1a, TaGlu1b, and TaGlu1c) were isolated from young wheat shoots. Although the TaGlu1s share very high sequence homology, the mRNA level of Taglu1c was much lower than the other two genes in 48-and 96-h-old wheat shoots. The expression ratio of each gene was different between two wheat cultivars. Recombinant TaGlu1b expressed in Escherichia coli was electrophoretically distinct fromTaGlu1a and TaGlu1c. Furthermore, coexpression of TaGlu1a and TaGlu1b gave seven bands on a native-PAGE gel, indicating the formation of both homo-and heterohexamers. One distinctive property of the wheat and rye glucosidases is that they function as hexamers but lose activity when dissociated into smaller oligomers or monomers. The crystal structure of hexameric TaGlu1b was determined at a resolution of 1.8 Å . The N-terminal region was located at the dimer-dimer interface and plays a crucial role in hexamer formation. Mutational analyses revealed that the aromatic side chain at position 378, which is located at the entrance to the catalytic center, plays an important role in substrate binding. Additionally, serine-464 and leucine-465 of TaGlu1a were shown to be critical in the relative specificity for DIMBOA-glucose (2-O-b-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-glucose (7-demethoxy-DIMBOA-glucose).b-D-Glucosidase (EC. 3.2.1.21) is a major member of the family GH1 and GH3 glycoside hydrolases and is responsible for hydrolysis of terminal non-reducing b-D-Glc residues in oligosaccharides (or polysaccharides) or glucoconjugates. In plants, b-D-glucosidases are involved in various functions, including lignification (Dharmawardhana et al., 1995), regulation of the biological activity of cytokinins (Brzobohatý et al

Comparative Analysis of Benzoxazinoid Biosynthesis in Monocots and Dicots: Independent Recruitment of Stabilization and Activation Functions

“…The K m values for GDIBOA (5.2 mM) and dhurrin (4.8 mM) hydrolysis were determined with C. orientalis crude extract and fit with the data from the heterologously expressed enzymes (see Supplemental Figure 7 online). In the grasses, GDIMBOA is the most efficiently hydrolyzed benzoxazinoid (Babcock and Esen, 1994;Sue et al, 2006); only in rye are the catalytic data for GDIBOA similar to the values for GDIMBOA (Nikus et al, 2003;Sue et al, 2006). Hence, the benzoxazinoid b-GLU functions of Co-GLU and monocot BxGLUs are distinct.…”

“…Catalytic data for BxGLUs have been determined for maize (Cicek et al, 2000), wheat (Sue et al, 2006), and rye (Nikus et al, 2003;Sue et al, 2006). The grass enzymes display a range of K m values from 0.1 to 1.4 mM for the substrates.…”

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

“…A structural overlay of the template sequences created using the DALI server (20) was used to verify that template regions identified as similar to SFR2 loops were loop structures in the template. The highest scoring template candidates included the following: S. solfataricus PDB entry 1UWT (17) and R. serpentina PDB entry 4A3Y (18), and also included Thermosphaera aggregans PDB entry 1QVB, a hyperthermophilic ␤-glycosidase (21); Paenibacillus polymyxa PDB entry 2JIE, a ␤-glucosidase B in complex with 2-fluoroglucose (22); and Triticum aestivum PDB entry 2DGA, a ␤-D-glucosidase in complex with glucose (23).…”

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

“…Simplified phylogenetic tree of the amino acid sequences of eukaryotic GH1 proteins with known structures and those of rice and Arabidopsis GH1 gene products. The protein sequences of the eukaryotic proteins with known structures are marked with four-character PDB codes for one of their structures, including Trifolium repens cyanogenic b-glucosidase (1CBG; Barrett et al, 1995), Sinapsis alba myrosinase (1MYR; Burmeister et al, 1997), Zea mays ZmGlu1 b-glucosidase (1E1F; Czjzek et al, 2000), Sorghum bicolor Dhr1 dhurrinase (1V02; Verdoucq et al, 2004), Triticum aestivum b-glucosidase (2DGA; Sue et al, 2006), Rauvolfia serpentina strictosidine b-glucosidase (2JF6; Barleben et al, 2007), and Oryza sativa Os3BGlu7 (BGlu1) b-glucosidase (2RGL; Chuenchor et al, 2008) from plants, along with Brevicoryne brassicae myrosinase (1WCG; Husebye et al, 2005), Homo sapiens cytoplasmic (Klotho) b-glucosidase (2E9M; Hayashi et al, 2007), and Phanerochaete chrysosporium (2E3Z; Nijikken et al, 2007), while those encoded in the Arabidopsis and rice genomes are labeled with the systematic names given by Xu et al (2004) and Opassiri et al (2006), respectively. One or two example proteins from each plant are given for each of the eight clusters of genes shared by Arabidopsis (At) and rice (Os) and the Arabidopsis-specific clusters At I (b-glucosidases) and At II (myrosinases), with the number of Arabidopsis or rice enzymes in each cluster given in parentheses.…”

“…The similar structures with great diversity in substrate specificity make plant GH1 enzymes an ideal model system to investigate the structural basis of substrate specificity. To date, seven plant b-glucosidase structures have been reported, including three closely related chloroplastic enzymes from maize (Czjzek et al, 2000(Czjzek et al, , 2001, sorghum (Verdoucq et al, 2004), and wheat (Triticum aestivum; Sue et al, 2006), the cytoplasmic strictosidine b-glucosidase from Rauvolfia serpentine (Barleben et al, 2007), and the secreted enzymes white clover (Trifolium repens) cyanogenic b-glucosidase (Barrett et al, 1995), white mustard (Sinapsis alba) myrosinase (thioglucosidase; Burmeister et al, 1997), and rice Os3BGlu7 (BGlu1; Chuenchor et al, 2008). These enzymes hydrolyze substrates with a range of structures, but they cannot account for the full range of b-glucosidase substrates available in plants, and determining the structural differences that bring about substrate specificity differences in even closely related GH1 enzymes has proven tricky (Verdoucq et al, 2003(Verdoucq et al, , 2004Sue et al, 2006;Chuenchor et al, 2008).…”