Abstract
Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. 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 suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy left( {Delta {text{G}}_{{{text{NU}}}}^{{{text{H}}_{2} {text{O}}}} } right) of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.
Highlights
Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel
We propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and the low catalytic activity of the enzyme when expressed in E. coli for biofuel production
SDS-PAGE analysis of the induced culture pellet revealed an intense band around 38 kDa, which was consistent with the calculated molecular mass of AAR and was absent in the uninduced cells (Fig. 1B)
Summary
Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. NU mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Abbreviations AAR Acyl-acyl carrier protein reductase ADO Aldehyde-deformylating oxygenase ACP Acyl carrier protein CD Circular dichroism CoA Coenzyme A DLS Dynamic light scattering GdnHCl Guanidine hydrochloride GFC Gel filtration chromatography IPTG Isopropyl β-d-1-thiogalactopyranoside MS Mass spectrometry NADPH Nicotinamide adenine dinucleotide phosphate UHPLC Ultra-high performance liquid chromatography UV Ultra violet. High energy density and compatibility of the long-chain alk(a/e)ne with the existing engines and infrastructure furthers its demand as an effective and ideal candidate for drop-in b iofuels[2,3]
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