Purification, crystallization and preliminary X-ray analysis of inositol dehydrogenase (IDH) fromBacillus subtilis

Abstract

Inositol dehydrogenase (IDH) is an enzyme that catalyses the NAD + -dependent oxidation of myo-inositol to scyllo-inosose. The enzyme has been purified to homogeneity by means of Ni 2+ -affinity chromatography and was crystallized in both native and selenomethionine (SeMet) labelled forms using the microbatch method. SAD X-ray diffraction data were collected to 2.0 Å resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 Å resolution at the Canadian Light Source (CLS); this is the first reported anomalous diffraction experiment from the CLS. The crystals belong to space group I222 and contain one molecule per asymmetric unit.

Converting NAD-Specific Inositol Dehydrogenase to an Efficient NADP-Selective Catalyst, with a Surprising Twist

“…Expression and purification of protein for crystallization was described previously. 20 Purified protein stock was thawed and diluted 1:1 with 25 mM Tris−HCl, pH 8.0, to a concentration of 3.25 mg mL −1 and incubated with 10 mM of NADPH, on ice, for 30 min. Crystal screening was done using the microbatch technique against crystallization screens from Qiagen.…”

Preparation and Application of 13C-Labeled myo-Inositol to Identify New Catabolic Products in Inositol Metabolism in Lactobacillus casei

“…In bacteria, mI can be used as a sole carbon source. − The well-characterized catabolic pathway in Bacillus subtilis , encoded by the iol operon, produces an equimolar mixture of dihydroxyacetone phosphate, acetyl-CoA, and CO 2 in six steps from mI, beginning with oxidation of the hydroxyl on C-2. ,, The enzyme that catalyzes this first step, myo -inositol dehydrogenase, is known to have a broad substrate scope but is selective for oxidation of an axial hydroxyl group. − …”

“…We have studied the enzymes in inositol catabolism of two Gram-positive bacteria, B. subtilis , and Lactobacillus casei . These studies indicated that mI dehydrogenase (mIDH) catalyzes the first step in mI catabolism by selectively oxidizing the hydroxyl group at position 2 forming scyllo -inosose (Figure ).…”

“…2,21,22 The enzyme that catalyzes this first step, myo-inositol dehydrogenase, is known to have a broad substrate scope but is selective for oxidation of an axial hydroxyl group. 22−24 We have studied the enzymes in inositol catabolism of two Gram-positive bacteria, B. subtilis 23,24 and Lactobacillus casei. 25 These studies indicated that mI dehydrogenase (mIDH) catalyzes the first step in mI catabolism by selectively oxidizing the hydroxyl group at position 2 forming scyllo-inosose (Figure 2).…”