Structural and functional insights into a novel aldehyde deformylating oxygenase with enhanced efficiency for biofuel applications

Aldehyde deformylating oxygenase (ADO) plays a crucial role in hydrocarbon biosynthesis by converting C n fatty aldehydes into C n−1 alkanes, key components of biofuels. However, ADO's low catalytic efficiency and thermostability hinder its industrial application. In this study, we identified a novel ADO from Pseudomonas plecoglossicida ( Ps ADO) using the Enzyme Function Initiative‐Enzyme Similarity Tool (EFI‐EST). Ps ADO contains a novel loop motif with a disulfide bond that forms a new substrate tunnel, enhancing both thermostability and catalytic efficiency. Ps ADO exhibited a melting temperature ( T m ) of over 61 °C, significantly higher than that of Prochlorococcus marinus ADO ( Pm ADO, T m = 41 °C), indicating superior stability. Ps ADO achieved its highest alkane yield at 10% oxygen, with a k cat of 1.38 min −1 , 106 times higher than that of Pm ADO for tridecane formation. A hybrid reducing system, combining ferredoxin from Synechocystis sp. PCC6803 and ferredoxin–NADP + reductase from Escherichia coli , further enhanced Ps ADO's activity compared with traditional chemical systems (PMS/NADH). AlphaFold 3 and CaverDock studies revealed that deleting Ps ADO's extended loop reduced alkane production by up to 9.4‐fold, while the N47A variant reduced tridecane formation by 1.25‐fold, confirming the importance of these structural features for substrate access and stability. These findings highlight Ps ADO's potential for biofuel applications, particularly in the production of long‐chain alkanes for jet fuel. Ps ADO's improved stability and efficiency make it a promising candidate for industrial biotechnology and biofuel production, with further optimization potential through genetic and metabolic engineering.