Presenters: Katie Crowther ; Jessie Alvarez ; Samantha Nielsen
Authors: Katie Crowther, Jessie Alvarez, Samantha Nielsen, Natasha Hudson
Faculty Advisor: Elizabeth Pierce
Institution: Southern Utah University
Periplasmic aldehyde oxidoreductase (PaoABC) is an Escherichia coli enzyme that is used to detoxify small organic compounds. The crystal structure of PaoABC shows an active site that is much less closed in than active sites of similar enzymes from different organisms. We hypothesize that PaoABC’s more open active site affects substrate binding affinity, and that by mutating amino acids around the PaoABC active site to make it more like other enzymes in the family, we can change its substrate binding and increase its ability to catalyze oxidation of a wider range of compounds. In this project, weare developing a protocol to overexpress active PaoABC in E. coliand are making mutants of PaoABC. We have made three different plasmids containing the PaoABC gene: one with an N-terminal his-tag, one with a C-terminal his-tag, and one construct to express the native protein with no tag. All three constructs are expressed at high levels in E. coliwhen the plasmid’s T7-lac promoter is induced. The C-terminally-his-tagged protein does not bind to Ni-NTA resin. Both the native and N-terminally his-tagged protein were purified, and were found to lack cofactors necessary for activity. Our current efforts are focused on expression of active enzyme and constructing mutants of the N-terminally his-tagged and native PaoABC.