Publications

• Structural investigation of myo-inositol dehydrogenase from Bacillus subtilis: implications for catalytic mechanism and inositol dehydrogenase subfamily classification.

  van Straaten K.E., Zheng H., Palmer D.R., Sanders D.A.

  Inositol dehydrogenase from Bacillus subtilis (BsIDH) is a NAD+-dependent enzyme that catalyses the oxidation of the axial hydroxy group of myo-inositol to form scyllo-inosose. We have determined the crystal structures of wild-type BsIDH and of the inactive K97V mutant in apo-, holo- and ternary c ... >> More

  Inositol dehydrogenase from Bacillus subtilis (BsIDH) is a NAD+-dependent enzyme that catalyses the oxidation of the axial hydroxy group of myo-inositol to form scyllo-inosose. We have determined the crystal structures of wild-type BsIDH and of the inactive K97V mutant in apo-, holo- and ternary complexes with inositol and inosose. BsIDH is a tetramer, with a novel arrangement consisting of two long continuous β-sheets, formed from all four monomers, in which the two central strands are crossed over to form the core of the tetramer. Each subunit in the tetramer consists of two domains: an N-terminal Rossmann fold domain containing the cofactor-binding site, and a C-terminal domain containing the inositol-binding site. Structural analysis allowed us to determine residues important in cofactor and substrate binding. Lys97, Asp172 and His176 are the catalytic triad involved in the catalytic mechanism of BsIDH, similar to what has been proposed for related enzymes and short-chain dehydrogenases. Furthermore, a conformational change in the nicotinamide ring was observed in some ternary complexes, suggesting hydride transfer to the si-face of NAD+. Finally, comparison of the structure and sequence of BsIDH with other putative inositol dehydrogenases allowed us to differentiate these enzymes into four subfamilies based on six consensus sequence motifs defining the cofactor- and substrate-binding sites. << Less

  Biochem J 432:237-247(2010) [PubMed] [EuropePMC]

• Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

  Daniellou R., Zheng H., Langill D.M., Sanders D.A., Palmer D.R.

  The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic co ... >> More

  The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. << Less

  Biochemistry 46:7469-7477(2007) [PubMed] [EuropePMC]

• Identification of two scyllo-inositol dehydrogenases in Bacillus subtilis.

  Morinaga T., Ashida H., Yoshida K.

  scyllo-Inositol (SI) is a stereoisomer of inositol whose catabolism has not been characterized in bacteria. We found that Bacillus subtilis 168 was able to grow using SI as its sole carbon source and that this growth was dependent on a functional iol operon for catabolism of myo-inositol (MI; anot ... >> More

  scyllo-Inositol (SI) is a stereoisomer of inositol whose catabolism has not been characterized in bacteria. We found that Bacillus subtilis 168 was able to grow using SI as its sole carbon source and that this growth was dependent on a functional iol operon for catabolism of myo-inositol (MI; another inositol isomer, which is abundant in nature). Previous studies elucidated the MI catabolic pathway in B. subtilis as comprising multiple stepwise reactions catalysed by a series of Iol enzymes. The first step of the pathway converts MI to scyllo-inosose (SIS) and involves the MI dehydrogenase IolG. Since IolG does not act on SI, we suspected that there could be another enzyme converting SI into SIS, namely an SI dehydrogenase. Within the whole genome, seven genes paralogous to iolG have been identified and two of these, iolX and iolW (formerly known as yisS and yvaA, respectively), were selected as candidate genes for the putative SI dehydrogenase since they were both prominently expressed when B. subtilis was grown on medium containing SI. iolX and iolW were cloned in Escherichia coli and both were shown to encode a functional enzyme, revealing the two distinct SI dehydrogenases in B. subtilis. Since inactivation of iolX impaired growth with SI as the carbon source, IolX was identified as a catabolic enzyme required for SI catabolism and it was shown to be NAD(+) dependent. The physiological role of IolW remains unclear, but it may be capable of producing SI from SIS with NADPH oxidation. << Less

  Microbiology 156:1538-1546(2010) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Purification, crystallization and preliminary X-ray analysis of inositol dehydrogenase (IDH) from Bacillus subtilis.

  Van Straaten K.E., Hoffort A., Palmer D.R., Sanders D.A.

  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 th ... >> More

  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 A resolution from a SeMet-labelled crystal at the Advanced Photon Source (APS) and a MAD data set was collected to 1.75 A 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. << Less

  Acta Crystallogr Sect F Struct Biol Cryst Commun 64:98-101(2008) [PubMed] [EuropePMC]