Structural and biochemical characterisation of a novel metagenome-derived salicylaldehyde dehydrogenase from alpine soil
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 (SALDAP) from an alpine soil metagenome. Phylogenetic analysis revealed that SALDAP 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. SALDAP 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 SALDAP 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 SALDAP and Pseudomonas NahF revealed that SALDAP 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 SALDAP, 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.