Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli

Abstract

An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5alpha. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 +/- 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.

FIG. 1.

Effect of culture age on malate dehydrogenase (10⁻¹) (▿) and aldehyde dehydrogenase (acetaldehyde, ▵; propionaldehyde, ○) activities in crude cell extracts. Samples were removed at 1- or 2-h intervals for measuring cell densities (▪) and enzyme activities. DH5α(pGEM) was grown as described in Materials and Methods. All values represent the means of three measurements, and similar results were obtained in a duplicate experiment. OD, optical density.

FIG. 2.

Details of the multiple sequence alignment for AldB (E. coli) with aldehyde dehydrogenase sequences of AcoD (A. eutrophus), AldA (V. cholerae), ALDH1 (human liver cytosol, NP-000680) and ALDH2 (human liver mitochondria, NP-000681) (National Center for Biotechnology Information database; www.ncbi.nlm.nih.gov). Dashes (−) indicate the amino acids lying close to the adenosine ribose. Asterisks (*), colons (:), and periods (.) indicate residues with identity, strong similarity, and weak similarity, respectively. The alignment represents the region where all the sequences exhibit identities greater than 60%.

FIG. 3.

Scheme showing the reaction pathway for the aldehyde dehydrogenase-catalyzed oxidation of an aldehyde (31).

FIG. 4.

A comparison of the region from H156 to E210 of HALD2 (human liver mitochondria, aldehyde dehydrogenase, NP-000681) with the region from H158 to D212 of the 3D-structure model derived for AldB (E. coli). The overlapping structures show that K194 and R197 from AldB are located at positions resembling those of K192 and E195 from HALD2.