Kinetic, computational, and phylogenetic characterization of PA2125 from Pseudomonas aeruginosa PAO1: A hydroxybenzaldehyde dehydrogenase of the newly defined ALDH33 family

PA2125 from Pseudomonas aeruginosa PAO1 is annotated as an aldehyde dehydrogenase (ALDH), but its substrate specificity, catalytic properties, and phylogenetic family assignment had not been established. Here, we cloned the PA2125 gene, produced and purified the recombinant PA2125 protein, and combined extensive substrate screening and steady-state kinetic characterisation with molecular dockings, molecular dynamics simulations, and phylogenetic analysis. PA2125 preferred NAD⁺ over NADP⁺, and screening of 54 aldehydes revealed a narrow substrate range centred on substituted benzaldehydes, particularly hydroxybenzaldehydes. Gentisaldehyde (GSA; 2,5-dihydroxybenzaldehyde) was the preferred substrate, with a KM of 5.3 µM, a kcat of 192 s⁻¹, and a remarkably high kcat/KM (3.54 × 10⁷ M⁻¹ s⁻¹). 3-Hydroxybenzaldehyde and 3,5-dihydroxybenzaldehyde also showed low KM values but approximately sevenfold lower catalytic efficiencies. Initial-velocity patterns supported an ordered sequential steady-state Bi Bi mechanism in which NAD⁺ binds first. Whereas GSA showed no substrate inhibition at concentrations up to 45 × KM, the other two hydroxybenzaldehydes showed pronounced total substrate inhibition-likely due to aldehyde binding at the nucleotide-binding site-at concentrations close to their KM. Docking identified plausible interactions involving unusual active-site residues, whereas molecular dynamics simulations showed that GSA more frequently populated and more readily accessed catalytically competent geometries. Phylogenetic analyses defined a distinct bacterial ALDH family, here designated ALDH33, comprising three subfamilies, with PA2125 belonging to ALDH33A. Together, these results identify GSA as the preferred in vitro substrate of PA2125, support its biochemical assignment as a gentisaldehyde dehydrogenase, and provide a foundation for investigating its physiological role in aromatic-aldehyde metabolism.