Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli.
The AdhE protein of Escherichia coli is a homopolymer of 96-kDa subunits harboring three Fe(2+)-dependent catalytic functions: acetaldehyde-CoA dehydrogenase, alcohol dehydrogenase, and pyruvate formatelyase (PFL) deactivase. By negative staining electron microscopy, we determined a helical assembly of 20-60 subunits into rods of 45-120 nm in length. The subunit packing is widened along the helix axis when Fe2+ and NAD are present. Chymotrypsin dissects the AdhE polypeptide between Phe762 and Ser763, thereby retaining the alcohol dehydrogenase activity on the NH2-terminal core, but destroying all other activities. PFL deactivation, i.e. quenching of the glycyl radical in PFL by the AdhE protein, was examined with respect to cofactor involvements (Fe2+, NAD, and CoA). This process is coupled to NAD reduction and requires the intact CoA sulfhydryl group. Pyruvate and NADH are inhibitors that affect the steady-state level of the radical form of PFL in a reconstituted interconversion cycle. Studies of cell cultures found that PFL deactivation in situ is initiated at redox potentials of greater than or equal to +100 mV. Our results provide insights into the structure/function organization of the AdhE multienzyme and give a rationale for how its PFL radical quenching activity may be suppressed in situ to enable effective glucose fermentation.
Kessler
D
D
Institut für Biologische Chemie, Universität Heidelberg, Federal Republic of Germany.
Herth
W
W
Knappe
J
J
D000123:Acetyltransferases / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D002918:Chymotrypsin / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005609:Free Radicals; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008958:Models, Molecular; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation
D029968:Escherichia coli Proteins; D005609:Free Radicals; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D000426:Alcohol Dehydrogenase; C068383:adhE protein, E coli; D000445:Aldehyde Oxidoreductases; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D002918:Chymotrypsin