Inactive and temperature-sensitive folding mutants of aldehyde dehydrogenase of Escherichia coli

Authors

  • Ana Limón Department of Biochemistry, Faculty of Pharmacy, University of Barcelona, Spain
  • Ana Enjuanes Department of Biochemistry, Faculty of Pharmacy, University of Barcelona, Spain
  • Juan Aguilar Department of Biochemistry, Faculty of Pharmacy, University of Barcelona, Spain

Keywords:

Escherichia coli, folding-mutants, temperature-sensitive, aldehyde dehydrogenase, aldA gene mutations

Abstract

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°C were shifted to 30°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.

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Published

2010-03-17

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Section

Research Articles