Inactive and temperature-sensitive folding mutants of aldehyde dehydrogenase of Escherichia coli.
Aldehyde dehydrogenase, encoded by the aldA gene in Escherichia coli, is inactive in nitrosoguanidine induced mutant strain ECL40 and temperature-sensitive in spontaneous mutant strain JA104. Both mutants were proven, by complementation experiments, to have a functional aldA regulator and promoter. In spite of no immunodetection of the aldA product, its specific transcript was present in the mutant extracts. It was subsequently proven that the immunodetection of aldehyde dehydrogenase in these mutants required denaturation, revealing that cells lacking the enzyme activity had the inactive protein in their extracts. Thus, the mutations seemed to affect the protein conformation. The temperature-sensitive aldehyde dehydrogenase did not show, neither in vivo nor in vitro, a different thermal stability compared to the wild type enzyme. In this temperature-sensitive strain, the recovery of active aldehyde dehydrogenase, in the presence of rifampicin but not of chloramphenicol, when cells grown at 37 degrees C were shifted to 30 degrees C indicated that this mutation affected the folding process of the protein at the restrictive temperature. Sequencing of the two mutant aldA corresponding genes determined a single amino acid change of Pro to Leu at position 182 for strain ECL40, and of Val to Met at position 145 for strain JA104. These mutations were thought to possibly promote changes in the local flexibility in the first case, and to perturb the packing of residues by steric hindrance in the second case.
Limón
A
A
Department of Biochemistry, Faculty of Pharmacy, University of Barcelona, Spain.
Enjuanes
A
A
Aguilar
J
J
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002701:Chloramphenicol / Q000494:pharmacology; D004252:DNA Mutational Analysis; D004790:Enzyme Induction; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012293:Rifampin / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002701:Chloramphenicol; D000444:Aldehyde Dehydrogenase; D012293:Rifampin