Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases

Abstract

Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step.

Figure 1.

Scheme of the general reaction mechanism of ALDH. The rate-limiting step for the three main mammalian isozymes is indicated. For ALD1, it is coenzyme dissociation, or k₉; for ALDH2, deacylation, or k₇; and for ALDH3, hydride transfer, or k₅.

Figure 2.

(A) Models of the tertiary structure of PAD and ALD. (Left) Backbone representation of the subunits obtained by modeling PAD and ALD as indicated in Materials and Methods, and subunit “A” from the tetramers of ALDH2 (used as the template to generate the model for PAD) and E. coli medium-chain ALDH (YDCW) (used as the template to generate the model for ALD), and the dimer of ALDH3. (Right) Spacefill representation of the same subunits, showing the dimer–dimer interface area if these subunits were tetramers. Hydrophobic and hydrophilic residues are shown in gray and in purple, respectively. (B) Alignment of the sequence of the region of residue 146 of PAD with the corresponding region of the sequence of human ALDH2 and ALD. PAD possesses a region of six extra amino acids shown by a rectangle in the sequence alignment and indicated by an arrow in the model; that region is not present in the other enzymes aligned.

Figure 3.

C-terminal region of ALDHs from mammalian, yeast, and E. coli. Residues 487 and 475, which are important for the stabilization of the coenzyme binding domain, are shown in rectangles. The residue numbers are based on human class 2 numbering.

Figure 4.

Alignment of the 197 region of the human and E. coli ALDHs. It has been proposed that this region is important for the coenzyme binding. Residue 197 and its two adjacent residues are shown by a rectangle. Numbers are according to E. coli aldB numbering.