Single amino acid exchanges affect the substrate preference of an acetaldehyde dehydrogenase
Year: 2025
Type: article
Abstract: The enzyme family of aldehyde dehydrogenases (EC 1.2.1._) consists of NAD(P)-dependent enzymes oxidizing a wide range of aliphatic or aromatic aldehydes to the corresponding acids. They are present in all organisms and typically consist of homodimers or -tetramers of 45- to 60-kDa subunits. The family is currently organized into 24 subclasses (Aldh1-24) which contain most of the known enzymes from animals and plants (Brocker et al. 2013 ; Hou and Bartels 2015 ), while microbes contain many more aldehyde dehydrogenases that are mostly not yet affiliated with the established subclasses. The enzymes of this family oxidize aldehydes to organic acids and, depending on the individual enzymes, either exhibit broad substrate spectra or are specific for only few closely related aldehydes (Shortall et al. 2021 ). While aldehyde oxidation to the corresponding acid is usually thermodynamically irreversible, the aldehyde dehydrogenases family also includes reversible aldehyde dehydrogenases which interconvert aldehydes and activated acids, such as acyl-phosphates, -adenylates or -thioesters with CoA. Some aldehyde dehydrogenases also occur as fusion proteins with alcohol dehydrogenases (e.g., AdhE of E. coli and many other bacteria), proline dehydrogenases (e.g. PutA) or acid kinases (e.g. eukaryotic Δ−1-pyrroline-5-carboxylate synthetase) (Martinelli et al. 2012 ; Extance et al. 2013 ; Liu et al. 2017 ). The structures of aldehyde dehydrogenases are highly conserved and exhibit three major domains per subunit; the first one comprises the N-terminal half of the subunit and is involved in binding NAD(P) + , the following second domain is almost as large as the first one and contains the catalytic center, and finally, a small third domain at the C-terminus is involved in oligomerization (Liu et al. 1997 ; Shortall et al. 2021 ). All of these enzymes contain an essential Cys residue in the catalytic center which forms a thiohemiacetal bond with the incoming aldehyde. In most members of the family, the catalytic Cys is deprotonated via a bridging water molecule with a conserved Glu residue to expedite formation of the thiohemiacetal intermediate. This “activated” form of the aldehyde then reacts with a bound NAD(P) + cofactor, getting oxidized to a covalently bound thioester after hydride abstraction (Shortall et al. 2021 ). The irreversible aldehyde dehydrogenases then hydrolyze this thioester with the nearby water ligand, releasing the contained energy as heat, while the reversible enzymes convert the bound thioester to another high-energy intermediate, such as an acyl-phosphate (e.g., glyceraldehyde phosphate dehydrogenases or reverse-acting enzymes like glutamyl phosphate reductases) or a CoA-thioester (e.g., AdhE or Clostridium kluyveri aldehyde dehydrogenase) (Smith and Kaplan 1980 ; Sirover 1999 ). Further, highly conserved amino acids are involved in NAD(P) + binding and positioning, as well as in forming an entry channel for the substrates. The latter consists of a “mouth” region on the surface of the N-terminal domain, which opens a funnel-like cavity reaching down to the catalytic Cys as one of the “bottom” residues. Moreover, some “neck” residues flanking this channel on the way toward the active site have been suggested to affect the substrate specificity of the enzymes (Shortall et al. 2021 ).
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $4,190
Funder: Deutsche Forschungsgemeinschaft
Award: G611919748
OpenAlex ID: W4409806631