Product release and substrate entry of aldehyde deformylating oxygenase revealed by molecular dynamics simulations

Cyanobacteria can produce alkanes equivalent to diesel fuels through a two-step enzymatic process involving acyl-(acyl carrier protein) reductase (AAR) and aldehyde deformylating oxygenase (ADO), providing a potential renewable biofuel source. AAR binds to ADO for efficient delivery of an aldehyde substrate and they have been proposed to dissociate when the alkane product is released from the same site as the substrate entrance of ADO. However, the dynamics of the substrate and product in ADO during substrate entry and product release are poorly understood. Here, we performed molecular dynamics (MD) simulations of ADO in the presence of substrate or product. We found that while the aldehyde substrate remains close to the active center of ADO before catalysis, the alkane product can dynamically rotate within the hydrophobic tunnel inside ADO toward the product exit after catalysis. Furthermore, the parallel cascade selection (PaCS)-MD simulations of ADO and the AAR/ADO complex identified the locations of the substrate entrance and the multiple exits for product release on ADO. Strikingly, the PaCS-MD simulations revealed that the alkane product can be released from the exit different from the substrate entrance without dissociation of AAR. Based on these results, we propose a reaction model for efficient alkane production by the AAR/ADO complex in which aldehydes and alkanes are synthesized simultaneously while AAR and ADO remain bound, and the aldehyde substrate can be delivered to ADO immediately after alkane release. Our study will be useful in improving the efficiency of bioalkane production using AAR and ADO.