Comparative enzymology and rational engineering of aldehyde deformylating oxygenases toward medium‐chain aldehydes

Alkane fuel properties depend on carbon chain length, requiring technologies to produce alkanes with desired chain lengths. Aldehyde deformylating oxygenase (ADO), which converts aldehydes to alkanes, is a key enzyme for tailoring alkane production. However, comparative understanding of diverse natural ADOs is limited, and strategies for engineering highly active variants with altered chain-length preference, particularly toward medium-chain aldehydes, remain underdeveloped. We compared the activities and substrate specificities of 20 natural ADOs toward C18, C16, C14, and C12 aldehydes under identical conditions to identify a high-activity scaffold for rational engineering. The ADOs showed similar substrate preference profiles, with relatively high activity toward C16 and C14 aldehydes and lower activity toward C12 aldehyde, but their activity levels varied substantially. NpADO showed the highest activity toward all tested substrates and was selected as the scaffold for shifting its substrate preference toward C12 aldehyde relative to C16 aldehyde. Single NpADO mutants were designed using previous findings and computational modeling. V29Y, Q111Y, and A122P showed no detectable activity toward C16 aldehyde, indicating improved relative preference for C12 aldehyde. Y126T, V185W, Y126S, V185F, W179R, and Y126V showed more than 1.5-fold higher C12 aldehyde activity than V184F_SeADO, a benchmark mutant with high specificity toward C12 aldehyde. Comparison of calculated substrate affinity with experimental activity showed that calculated substrate affinity alone did not quantitatively account for ADO activity, suggesting that other structural or dynamic factors, including substrate positioning, may contribute to catalysis. These findings provide an experimental basis for natural ADO scaffold selection, engineering chain-length preference toward medium-chain aldehydes, and data-driven design of ADO catalysts for chain-length-tailored alkane production.