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      X-ray Crystallography of Inositol Dehydrogenase Enzymes

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      BERTWISTLE-DISSERTATION.pdf (169.7Mb)
      Date
      2015-04-30
      Author
      Bertwistle, Drew
      Type
      Thesis
      Degree Level
      Doctoral
      Metadata
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      Abstract
      Lactobacillus casei BL23 expresses two enzymes encoded by the genes iolG1 and iolG2. They have been putatively assigned as myo-inositol dehydrogenases by sequence comparison. The enzyme catalyzes the reversible conversion of myo-inositol to scyllo-inosose and the concurrent reduction of NAD+ to NADH. iolG1 was subsequently determined to be a myo-inositol dehydrogenase but iolG2 was determined to be a scyllo-inositol dehydrogenase. Sequence analysis and kinetics by themselves did not provide insight as to why the enzymes are functionally different. This manuscript provides a structural rationalization for the differences in stereoisomer selectivity by X- ray crystal structure analysis and comparison. High resolution apo, binary, and ternary crystal structures for iolG1 and iolG2 wild type enzymes were determined. For iolG1 the ternary structures were determined for myo-inositol and d-chiro-inositol and for iolG2 the scyllo-inositol bound structure was determined. The high resolution structure information revealed the composition of their respective active sites and showed that subtle differences in critical amino acids for each enzyme define the orientation of the inositol stereoisomer for inline transfer of a hydride to NAD+. Mutagenesis studies of a closely related myo-inositol dehydrogenase from Bacillus subtilis were carried out. The wild type structure for BsIDH had already been determined and characterized. A portion of the results in this manuscript briefly explore structures of dehydrogenase mutants which validate the structural role of residues involved in cofactor selectivity
      Degree
      Doctor of Philosophy (Ph.D.)
      Department
      Physics and Engineering Physics
      Program
      Physics
      Supervisor
      Sanders, David; Bergstrom, Jack
      Committee
      Tse, John; Chapman, Dean; Palmer, David; Saridakis, Vivian; Smolyakov, Andrei
      Copyright Date
      April 2015
      URI
      http://hdl.handle.net/10388/ETD-2015-04-2027
      Subject
      X-ray Crystallography
      myo-inositol dehydrogenase
      scyllo-inositol dehydrogenase
      structural biology
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