Background: Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in E. coli, leading to varying alkane levels. Intriguingly, the structural characterization remains largely elusive as AAR alone failed to form stable crystals, possibly due to a number of intrinsically flexible random regions. The present work attempts to fill a gap in the literature by investigating the crucial structural aspects of AAR protein associated with its stability and folding.
Results: The AAR protein was recombinant expressed in E. coli and purified by metal affinity and gel filtration chromatography. Characterization by dynamic light scattering experiment revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles in the range of 36.4 to 51.6 nm. Intact mass spectrometry revealed that recombinant AAR was heterogenous due to diverse lipidation and de-lipidation resulted in a single mass peak of 40296.87 Da as predicted. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution. Lower concentration of GdnHCl appeared to be stabilizing the protein, suggesting that the protein milieu plays a significant role in dictating it’s folding. Standard free energy (∆GH2ONU) of ~4.5 kcal/mol for steady-state unfolding of AAR indicated borderline stability of the protein. Molecular dynamics simulation conducted on AAR structure in presence of KCl, an ionic solvent with similar properties as GdnHCl at lower concentrations, suggested that KCl mediates structural stabilization especially at the concentration of 375 mM, and thus was responsible for enhancing its activity. KCl presence also resulted in regional alteration towards the binding site of its neighbouring pathway enzyme, ADO, thus paving the way for coordinated catalysis.
Conclusion: Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show path for building superior biocatalyst for higher biofuel production.