title: Structural and functional impairment of an Old Yellow Enzyme homologue upon affinity tag incorporation.
abstract: Recently, it has been reported that the previously uncharacterized YqjM protein from Bacillus subtilis is a true homologue of the physiologically enigmatic yeast Old Yellow Enzyme (OYE). In this study, it was also demonstrated that YqjM is involved in the oxidative stress response of B. subtilis, thus highlighting a novel direction to pursue the role of the OYE family of proteins in the cell. As part of an attempt to pin down the exact physiological role of these enzymes, both a N-terminal glutathione S-transferase and a C-terminal histidine-tagged form of the protein were created to enable "pull-down" assays and identify interacting partners which could aid in the functional definition. However, here we report on a comparison of the biochemical properties of the tagged forms with the native/untagged YqjM, revealing critical differences in the catalytic activities and quaternary structure of the protein forms. UV-visible spectrophotometric features as well as steady state and individual half-reaction kinetic parameters show that the affinity tagged forms are severely impaired both in ligand binding and catalysis. Gel filtration and dynamic light scattering studies show that incorporation of a tag also has major effects on the quaternary structure of the protein by disrupting the native tetrameric conformation which may help to explain the observed differences. The study thus highlights important considerations for expression construct design when isolating members of the OYE family of proteins.
authors.affiliation: ETH-Zürich, Institut für Pflanzenwissenschaften, Universitätstr. 2, CH-8092 Zürich, Switzerland. teresa.fitzpatrick@ipw.biol.ethz.ch
authors.forename: Teresa B
authors.initials: TB
authors.lastname: Fitzpatrick
authors.forename: Sigrid
authors.initials: S
authors.lastname: Auweter
authors.forename: Karina
authors.initials: K
authors.lastname: Kitzing
authors.forename: Tim
authors.initials: T
authors.lastname: Clausen
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Amrhein
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D002850:Chromatography, Gel; D015870:Gene Expression; D009252:NADPH Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018384:Oxidative Stress / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012542:Scattering, Radiation; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009252:NADPH Dehydrogenase