X-ray Crystallography of Inositol Dehydrogenase Enzymes

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

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Physics

Grantor

University of Saskatchewan

Year dc:date.issued

2015

Author dc:creator

• Bertwistle, Drew

Advisors dc:contributor.advisor

• Sanders, David
• Bergstrom, Jack

Committee members dc:contributor.committeemember

• Tse, John
• Chapman, Dean
• Palmer, David
• Saridakis, Vivian
• Smolyakov, Andrei

• X-ray Crystallography
• myo-inositol dehydrogenase
• scyllo-inositol dehydrogenase
• structural biology

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/10388/ETD-2015-04-2027

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/ETD-2015-04-2027