A Novel Salicylaldehyde Dehydrogenase from Alpine Soil Metagenomes Reveals a Unique Catalytic Mechanism

Metagenomic approaches have revolutionised the discovery of novel enzymes with biotechnological potential from unexplored environments. Here, we report the identification and comprehensive characterisation of a novel salicylaldehyde dehydrogenase (SALD _AP ) from an alpine soil metagenome. Phylogenetic analysis revealed that SALD _AP is the first experimentally characterised alphaproteobacterial SALD, forming a distinct evolutionary clade among known bacterial SALDs. The recombinant enzyme showed strict specificity for NAD⁺ and exceptional catalytic efficiency toward aromatic aldehydes, with benzaldehyde as the preferred substrate. SALD _AP was most active under mildly alkaline conditions (optimum pH 8.0) and tolerated a range of chemical environments, though high concentrations of certain metal ions and solvents were inhibitory. Kinetic analysis demonstrated that SALD _AP binds and oxidises aromatic substrates much more efficiently than aliphatic aldehydes, with catalytic efficiencies exceeding 10⁶ M⁻¹ s⁻¹ for aromatics. The enzyme was stabilised by the simultaneous presence of substrate and cofactor, as shown by differential scanning fluorimetry. Molecular docking with the crystal structures of SALD _AP and Pseudomonas NahF revealed that SALD _AP utilises a unique arrangement of active site residues (ASN-137, ARG-145, GLU-238, and CYS-272) to mediate catalysis. Based on structural and docking data, we propose a distinct catalytic mechanism for SALD _AP , in which ASN-137 plays a central role in substrate binding and stabilisation. This study expands the functional diversity of the ALDH superfamily. It establishes a new paradigm for aromatic aldehyde oxidation, providing valuable insights for the engineering of ALDHs for environmental bioremediation and synthetic biology applications.