Kinetic and structural analysis of human ALDH9A1

Abstract

Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes, which detoxify aldehydes produced in various metabolic pathways to the corresponding carboxylic acids. Among the 19 human ALDHs, the cytosolic ALDH9A1 has so far never been fully enzymatically characterized and its structure is still unknown. Here, we report complete molecular and kinetic properties of human ALDH9A1 as well as three crystal forms at 2.3, 2.9, and 2.5 Å resolution. We show that ALDH9A1 exhibits wide substrate specificity to aminoaldehydes, aliphatic and aromatic aldehydes with a clear preference for γ-trimethylaminobutyraldehyde (TMABAL). The structure of ALDH9A1 reveals that the enzyme assembles as a tetramer. Each ALDH monomer displays a typical ALDHs fold composed of an oligomerization domain, a coenzyme domain, a catalytic domain, and an inter-domain linker highly conserved in amino-acid sequence and folding. Nonetheless, structural comparison reveals a position and a fold of the inter-domain linker of ALDH9A1 never observed in any other ALDH so far. This unique difference is not compatible with the presence of a bound substrate and a large conformational rearrangement of the linker up to 30 Å has to occur to allow the access of the substrate channel. Moreover, the αβE region consisting of an α-helix and a β-strand of the coenzyme domain at the dimer interface are disordered, likely due to the loss of interactions with the inter-domain linker, which leads to incomplete β-nicotinamide adenine dinucleotide (NAD+) binding pocket.

Rare genetic variants affecting urine metabolite levels link population variation to inborn errors of metabolism

“…The maximum number of qualifying variants was 25 for HAL (total minor allele count [MAC] of 188, p-burden = 1.5e−11 for trans-urocanate), and the highest total MAC was 320 for 12 variants in ALDH9A1 (p-SKAT = 7.5e−29 for X-24807, a yet unnamed metabolite) (Supplementary Data 3). Aldehyde dehydrogenases detoxify aldehydes from different metabolic reactions to the corresponding carboxylic acids, with ALDH9A1 showing wide substrate specificity to amino-, aliphatic and aromatic aldehydes 23 . Additional information about the unknown molecules in Table 1 can be obtained from mzML files available through the EMBL-EBI database Metabolights (Data Availability).…”

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium

“…It is known that Gm BADH, Pa BADH, Sa BADH, and Hs ALDH9A1 have an α/β structure with a Rossman‐type folding. The catalytic domain is formed by six parallel β‐sheets that, when interacting with NAD + , a rearrangement may occur facilitating the protein denaturation 27‐30 . Therefore, we propose that the pkBADH‐NAD + complex is more thermolabile in the absence of K + .…”

“…Končitíková et al proposed that the Hs ALDH9A1 inter‐domain linker (residues 449‐SPVELPFGGYKKSGFGRENG–470) does not adopt the typical folding observed in all known ALDHs structures 29 . Those authors proposed that the interdomain linker adopts a new position and folding, and a drastic rearrangement up to 30 Å is required to the NAD + access the substrate channel 29 . Interestingly, that sequence is present in the pkBADH (Figure 1), and our CD data showed a 19% increase in the pkBADH α‐helical structure when pkBADH binds the NAD + (Table 1).…”

“…Recently, the crystal structure of the human ALDH9A1 enzyme was reported, in which differences in the coenzyme domain (residues 232‐256) and in the interdomain linker (residues 449‐470), do not adopt the typical fold so far observed in all known ALDHs' structures 29 . That highly conserved region has an important role both in stabilizing the coenzyme binding site and in interacting with the bound substrate.…”

“…Indeed, it protrudes alongside the edge between the coenzyme and the catalytic domains and establishes several H‐bonds, including the βE strand forming the coenzyme binding site. In the ALDH9A1 structure, this interdomain region adopts a new position, and the fold is associated with substantial conformational changes compared with other ALDH's structure 29 …”