Molecular characterization of PadA, a phenylacetaldehyde dehydrogenase from Escherichia coli

Abstract

The padA gene encoding the phenylacetaldehyde dehydrogenase involved in the catabolism of 2-phenylethylamine in Escherichia coli has been cloned, sequenced, and located at 31.0 min on the chromosome. The deduced PadA polypeptide contains 499 amino acid residues with a predicted molecular mass of 53.7 kDa, and its primary structure reveals significant similarity with that of members of the aldehyde dehydrogenase superfamily. By engineering optimal transcription and translation elements, a high expression of the padA gene has been achieved. The active PadA enzyme is a homodimer that prefers NAD+ over NADP+ as coenzyme. The enzyme efficiently oxidizes only phenylacetaldehyde-like aromatic aldehydes, and has a weak esterase activity with /7-nitrophenol. The padA gene constitutes a new catabolic tool for designing DNA cassettes to expand the abilities of microorganisms to degrade toxic aromatic compounds.© 1997 Federation of European Biochemical Societies.

Biodegradation of Aromatic Compounds by Escherichia coli

“…Although many of the aldehyde dehydrogenases so far characterized are homotetramers, the quaternary structure in this superfamily of enzymes is evolutionarily variable, and several homodimers, such as the HpaE (HpcC) and Sad aldehyde dehydrogenases from the HPC dehydrogenative route of E. coli (see above), have also been described. As already reported for the PAL dehydrogenase from Achromobacter eurydice (101), a high concentration of PAL inhibited the PadA (FeaB) enzyme (95,127). Substrate inhibition has also been observed in other aldehyde dehydrogenases (140).…”

“…The padA gene is transcribed in the opposite direction to that of maoA and maoB (Fig. 6A), indicating that these three genes, although involved in the same metabolic pathway and physically associated, do not constitute an operon (95). Since these genes map immediately adjacent to the left end of the paa cluster (Figs.…”

“…The maoA (tynA) gene encoding the monoamine oxidase of E. coli K-12 (49) and W (95) has been cloned and overexpressed (13,95,299). The periplasmic MaoA protein (757 aa) shows 84% amino acid sequence identity to the equivalent soluble monoamine oxidase from Klebsiella aerogenes, although the similarity to amino oxidases of gram-positive bacteria and eukaryotic organisms is much lower (124).…”

“…All the conserved motifs that characterize the superfamily of aldehyde dehydrogenases (237) are present in the primary structure of PadA. The PadA protein was partially purified and was shown to be an homodimer (95). Although many of the aldehyde dehydrogenases so far characterized are homotetramers, the quaternary structure in this superfamily of enzymes is evolutionarily variable, and several homodimers, such as the HpaE (HpcC) and Sad aldehyde dehydrogenases from the HPC dehydrogenative route of E. coli (see above), have also been described.…”

“…Although the affinity of PadA for NAD ϩ was 2 orders of magnitude lower than that for PAL (K m , 3 M), this may be compensated for the high concentration (806 M) of the NAD ϩ /NADH pool in E. coli. On the other hand, since poor expression of the padA gene can be predicted based on its codon usage, the high affinity of PadA for PAL might consti-tute an effective safety mechanism to eliminate free aldehyde that could be toxic to the cell (95).…”