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

Metagenomic approaches have revolutionised the discovery of novel enzymes with ecological and biotechnological significance from different environments. Here, we report the comprehensive characterisation of a novel salicylaldehyde dehydrogenase (SALD_AP) obtained 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 enzymes. The recombinant enzyme exhibited strict specificity for NAD⁺ and exceptional catalytic efficiency toward aromatic aldehydes, with benzaldehyde as the preferred substrate. Kinetic analyses showed catalytic efficiencies exceeding 10⁶ M⁻¹ s⁻¹ for aromatics, whereas aliphatics were oxidised with much lower efficiency, consistent with ecological specialisation for aromatic catabolism in alpine soils enriched in lignin-derived compounds. 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 metals and solvents were inhibitory. Differential scanning fluorimetry demonstrated that the enzyme was stabilised by ligand binding, with maximal thermal stability observed when both substrate and cofactor were present. Structural alignment with Pseudomonas NahF and docking analyses revealed that SALD_AP employs a distinctive catalytic configuration involving ASN-137, ARG-145, GLU-238, and CYS-272, highlighting a non-canonical role for ASN-137 in substrate binding and stabilisation. Based on these findings, we propose a mechanistic model for SALD_AP that expands the catalytic diversity of the aldehyde dehydrogenase superfamily. This study establishes a new paradigm for aromatic aldehyde oxidation, underscores the ecological significance of SALD_AP in alpine soil microbiomes, and provides a foundation for engineering novel biocatalysts for bioremediation and synthetic biology applications.