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Isomaltase from Saccharomyces cerevisiae is an oligo-1,6-glucosidase that preferentially hydrolyzes isomaltose, with little activity towards isomaltotriose or longer oligosaccharides. An amino-acid sequence analysis of the isomaltase revealed that it belongs to glucoside hydrolase family 13. Recombinant isomaltase was purified and crystallized by the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant. The crystals belonged to space group C2, with unit-cell parameters a = 95.67, b = 115.42, c = 61.77 Å, β = 91.17°. X-ray diffraction data were collected to 1.35 Å resolution from a single crystal on a synchrotron-radiation source.
α-Glucosidases hydrolyze the terminal nonreducing α-d-glucosidic linkage of disaccharides, oligosaccharides and polysaccharides, with the release of α-glucose. The substrate-specificity of α-glucosidases differs greatly depending on the source of the enzyme. Most α-glucosidases (EC 18.104.22.168; α-d-glucoside glucohydrolases) preferentially hydrolyze maltose (Needleman et al., 1978 ), whereas another class of α-glucosidases, oligo-1,6-glucosidases (EC 22.214.171.124; oligosaccharide oligo-1,6-glucohydrolases), act on the α-1,6-glucosidic linkage of isomaltooligosaccharides and dextran (Linder & Sund, 1981 ; Suzuki et al., 1982 ).
α-Glucosidase is classified into glucoside hydrolase (GH) family 13 on the basis of its amino-acid sequence relationship to other glycosyl hydrolases. Many primary structures of members of GH family 13 from various origins are now available and have been compared with each other. The existence of four highly conserved regions (regions I–IV) and three acidic residues located in the conserved regions as catalytic residues have been reported (Matsuura et al., 1984 ; Nakajima et al., 1986 ; Svensson, 1988 ). Furthermore, all GH family 13 enzymes for which three-dimensional structures have been solved have a common multidomain structure composed of three domains: A, B and C (MacGregor et al., 2001 ). Domain A is a catalytic domain containing a (β/α)8-barrel. Domain B is found inserted between the third β-sheet and the helix of the (β/α)8-barrel and may play a role in both enzyme stability and substrate binding. Domain C has a β-barrel structure of eight antiparallel β-strands in a double Greek-key motif. The catalytic nucleophile in the conserved region II is located in the loop extending from β-strand 4 and the general acid–base catalyst in the conserved region III is located at the C-terminus of β-strand 5.
Saccharomyces cerevisiae contains two α-glucosidases, namely α-1,4-glucosidase (maltase) and oligo-1,6-glucosidase (isomaltase), which preferentially hydrolyze maltose or isomaltose and methyl α-d-glucopyroside (α-mg), respectively. The expression of these enzymes is independently controlled by different polymeric genes, namely MAL and MGL (Carlson, 1987 ; Vanoni et al., 1989 ; Johnson & Carlson, 1992 ). Maltase (the MAL6 product of S. cerevisiae) preferentially hydrolyzes maltose but not isomaltose or α-mg, whereas isomaltase hydrolyzes isomaltose and α-mg but not maltose (Khan & Eaton, 1967 ; Needleman et al., 1978 ).
Isomaltase from S. cerevisiae shows a notable substrate-specificity. Generally, oligo-1,6-glucosidases preferentially hydrolyze isomaltotriose and show high activity toward isomaltooligosaccharides or dextran (Linder & Sund, 1981 ; Suzuki et al., 1982 ; Russell & Ferretti, 1990 ; Saburi et al., 2006 ). However, isomaltase from S. cerevisiae shows highest activity toward isomaltose and little activity toward isomaltotriose and isomaltotetraose.
In 1997, the crystal structure of oligo-1,6-glucosidase from Bacillus cereus was solved at 2.0 Å resolution (Watanabe et al., 1997 ). Recently, the structures of dextran glucosidase in an uncomplexed form and of its mutant in complex with isomaltotriose at 2.2 Å resolution have been determined (Hondoh et al., 2008 ). However, in order to obtain a better understanding of the structure–function relationship of oligo-1,6-glucosidases, more precise structures are required. In the present work, we report the crystallization of S. cerevisiae isomaltase and present a preliminary crystallographic analysis of the crystals obtained.
The cloning and expression of the isomaltase gene in Escherichia coli have been described previously (Yamamoto et al., 2004 ). A transformant of E. coli containing the isomaltase expression plasmid was cultured in 150 ml Luria–Bertani medium (1% tryptone, 0.5% yeast extract, 1% NaCl) containing 50 µg ml−1 ampicillin at 310 K. Isopropyl β-d-1-thiogalactopyranoside was added (to a final concentration of 0.02 mM) when the optical density at 660 nm of the culture reached 0.6 and the culture was incubated for a further 12 h. E. coli cells were resuspended in 10 mM sodium phosphate buffer pH 6.8 and sonicated. The cell-free extract was applied onto a QAE-Toyopearl 650M column (Toyo Soda, Japan) equilibrated with 20 mM sodium phosphate buffer pH 6.8 and washed with the same buffer. The enzyme was eluted with a linear gradient of 0–150 mM NaCl in the same buffer. Active fractions were pooled and dialyzed against the same buffer and then subjected to chromatography on hydroxyapatite (Micro-Prep Ceramic Hydroxyapatite Type I; BioRad). The purified enzyme was eluted with a linear gradient of 20–200 mM sodium phosphate buffer pH 6.8.
Prior to crystallization, the isomaltase was dialyzed against 5 mM HEPES buffer pH 7.3 and concentrated to 4.5 mg ml−1 by ultrafiltration with a Centricon YM-10 filter (Millipore, USA). Hampton Research kits were used to determine the initial crystallization conditions. Drops consisting of 3 µl protein solution and 3 µl reservoir solution were equilibrated over 0.5 ml reservoir solution using the hanging-drop vapour-diffusion method. Crystals were obtained using solution No. 24 [0.2 M lithium acetate dehydrate, 20%(w/v) polyethylene glycol (PEG) 3350] of the PEG/Ion Screen at 293 K. The pH of the buffer solution and the concentration of PEG were optimized and the temperature was varied. After optimization, rod-shaped crystals grew to maximum dimensions of 0.2 × 0.2 × 1.5 mm in two weeks (Fig. 1 ). The reservoir solution consisted of 50 mM HEPES pH 7.3, 0.2 M lithium acetate and 19%(w/v) PEG 3350. The crystallization temperature was 288 K.
A single crystal was transferred from the crystallization drop to a cryoprotectant solution containing 50 mM HEPES pH 7.3, 0.2 M lithium acetate, 19%(w/v) PEG 3350 and 10%(v/v) glycerol for a few seconds. The crystal was then flash-cooled to 100 K in a nitrogen-gas stream in order to prevent radiation damage during data collection. A data set was collected from a single crystal using synchrotron radiation of wavelength 0.9000 Å on beamline BL44XU of SPring-8 (Hyogo, Japan) with an imaging-plate X-ray detector system (DIP6040). A total of 200 images were collected with 1° oscillation and an exposure time of 5 s per image. Data processing was performed with HKL-2000 (Otwinowski & Minor, 1997 ).
The isomaltase crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 95.67, b = 115.42, c = 61.77 Å, β = 91.17°. Detailed data-collection statistics are summarized in Table 1 . The total of 615 250 measured reflections were reduced to 146 713 unique reflections with an overall R merge of 6.6%. This represents 99.8% completeness at 1.35 Å resolution. The outermost shell of resolution between 1.40 and 1.35 Å was 99.5% complete with an R merge of 12.2%.
Assuming the presence of one molecule in the asymmetric unit, the Matthews parameter (V M; Matthews, 1968 ) of 2.5 Å3 Da−1 (50.1% solvent content by volume) is well within the allowed range for crystalline proteins.
Structural determination is in progress using molecular replacement with the reported structure of the oligo-1,6-glucosidase from B. cereus (PDB code 1uok; Watanabe et al., 1997 ) as a search model.