The operon contains seven genes (encodes a neopullanase, which hydrolyzes the -1,4 linkages in starch to produce smaller oligosaccharides; encodes an -glucosidase, which is able to break down these smaller oligomers into glucose; encode outer membrane proteins involved in starch binding; and?encodes a protein, the role of which is unclear, but which has high sequence similarity to amylases (D’Elia and Salyers, 1996; Reeves et?al., 1997). Carbohydrate active enzymes have been classified into families based upon their amino acid sequence similarities (Coutinho and Henrissat, 1999). the host (Comstock and Coyne, 2003; Xu et?al., 2003; Zocco et?al., 2007). Perhaps the most well-characterized polysaccharide utilization machinery in is that encoded by the operon. The operon contains seven genes Osthole (encodes a neopullanase, which hydrolyzes the -1,4 linkages in starch to produce smaller oligosaccharides; encodes an -glucosidase, which is able to break down these smaller oligomers into glucose; encode outer membrane proteins involved in starch binding; Osthole and?encodes a protein, the role of which is unclear, but which has high sequence similarity to amylases (D’Elia and Salyers, 1996; Reeves et?al., 1997). Carbohydrate active enzymes have been classified into families based upon their amino acid sequence similarities (Coutinho and Henrissat, 1999). Currently there are 113 sequence-distinct glycosidase families. A feature of almost all classical CAZy families is that, since sequence dictates structure, and structure determines function, the catalytic mechanism is conserved within a sequence-based family (Henrissat and Davies, 1997). Exceptions to this rule are rare and unusual: GH4 and GH109 enzymes are not classical hydrolases, but instead use NAD+ in a transient reduction/oxidation reaction with leaving group elimination (Rajan et?al., 2004), and GH23 is a family with both inverting and retaining hexosaminidases, but the catalytic mechanism of neither is understood, and may involve substrate Osthole participation in catalysis. The -glucosidase encoded by the gene belongs to family GH97 (Hughes et?al., 2003) of this Carbohydrate Active enZymes (CAZy) classification (http://www.cazy.org/), the catalytic mechanism of which was unknown prior to this work, but had been predicted to be retaining in an insightful bioinformatics analysis (Naumoff, 2005). possesses a total of 10 GH97 members, and there are currently 69 other bacterial GH97 open reading frames, and one from the archaea (-galactosidase (Smith and Salyers, 1991) and an enzyme from (Hughes et?al., 2003) have been investigated previously, and both have been shown to possess -glucosidase activity. Representatives Inverting subfamily sequences are shown in blue and retaining subfamily sequences in red. The inverting-retaining transition reflects a loss of the inverting base?and its replacement by a Gly-Asp shift elsewhere in the sequence. GH97 has six unusual sequences, which appear to contain neither inverting nor retaining catalytic signatures; the outlier is shown in green. The full annotated family tree is given in Supplemental Data. Mechanistic Studies on a Second GH97 Enzyme To ascertain whether an enzyme that possessed the opposite set of motifs to (alone possesses 10 GH97 enzymes, it suggests that there may be further substrate diversity to discover within this fascinating family. Each of the enzymes was shown to be extremely specific for either glucose- (-galactosidase (-galactosidase. The presence of interactions between the calcium ion and four?glutamate residues in VPI-5482 genomic DNA using primers Rabbit Polyclonal to C56D2 that gave ligation-independent cloning (LIC) compatible ends. These were ligated into an LIC-modified pET28a vector using standard procedures (Bonsor et?al., 2006). Protein production and purification was identical for each protein. Plasmid containing the gene of interest was transformed into BL21 (DE3) cells, and cultured in 0.5 L autoinduction media (Studier, 2005) supplemented with 50 g ml?1 kanamycin at 37C for 8 hr. Protein production was induced Osthole at 30C overnight. Cells were harvested and resuspended in 20 mM HEPES, pH 7, 150 mM NaCl, and lysed by sonication. The supernatant was applied to a 5?ml HisTrap nickel-Sepharose column (GE Healthcare), preequilibrated in the same buffer, and the protein eluted from an imidazole gradient. The protein was dialyzed to remove the imidazole, concentrated, and purified further on an?S200 16/60 gel filtration column, preequilibrated in 20 mM HEPES, pH 7, 150?mM NaCl. Selenomethionine-containing BtGH97a was obtained and purified in the same way as described for the native protein, except that the autoinduction media contained selenomethionine (SeMet). Crystallization BtGH97a, at 10 mg ml?1, was crystallized from 18%C22% polyethylene glycol 3350 and 0.02 M sodium/potassium phosphate. Crystals were cryoprotected in a solution containing the relevant mother liquor with the addition of 25% ethylene glycol, and were flash frozen in liquid nitrogen. Crystals were also grown in the presence of 2.5 mM 1 or 2 2 using the same crystallization conditions. Data Collection, Structure Solution,.