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 (PsADO) using the Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST). PsADO contains a novel loop motif with a disulfide bond that forms a new substrate tunnel, enhancing both thermostability and catalytic efficiency. PsADO exhibited a melting temperature (T_m) of over 61 °C, significantly higher than that of Prochlorococcus marinus ADO (PmADO, T_m = 41 °C), indicating superior stability. PsADO achieved its highest alkane yield at 10% oxygen, with a k_cat of 1.38 min^-1, 106 times higher than that of PmADO for tridecane formation. A hybrid reducing system, combining ferredoxin from Synechocystis sp. PCC6803 and ferredoxin-NADP⁺ reductase from Escherichia coli, further enhanced PsADO's activity compared with traditional chemical systems (PMS/NADH). AlphaFold 3 and CaverDock studies revealed that deleting PsADO'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 PsADO's potential for biofuel applications, particularly in the production of long-chain alkanes for jet fuel. PsADO'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.