title: Inactivation of E. coli pyruvate formate-lyase: role of AdhE and small molecules.
abstract: Escherichia coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectable, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both beta-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59718, USA.
authors.forename: Mbako R
authors.initials: MR
authors.lastname: Nnyepi
authors.forename: Yi
authors.initials: Y
authors.lastname: Peng
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry*; D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes / Q000737:chemistry*
chemical_list: D004798:Enzymes; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D000426:Alcohol Dehydrogenase; C068383:adhE protein, E coli; D000445:Aldehyde Oxidoreductases; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme; C022560:formate C-acetyltransferase