Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

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 $$\left( {\Delta {\text{G}}_{{{\text{NU}}}}^{{{\text{H}}_{2} {\text{O}}}} } \right)$$ Δ G NU H 2 O 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.

Macrocyclic Cavitand β‐Cyclodextrin Inhibits the Alcohol‐Induced Trypsin Aggregation

“…During fibrillation, Trypsin unfolds and the subsequently forms fibrils where the probe, tryptophan, is experiencing different microenvironments in its native and aggregated states [26] . In the native state, the tryptophan amino acid residue(s) experience a hydrophobic environment, and hence its inherent fluorescence remains quenched, a condition referred to as “native quenching” [27] . However, in the presence of 60 % EtOH, Trypsin gets denatured and the active intrinsic fluorophores experience a more hydrophilic environment and the native quenching is lost.…”

“…However, in the presence of 60 % EtOH, Trypsin gets denatured and the active intrinsic fluorophores experience a more hydrophilic environment and the native quenching is lost. This results in the rise in fluorescence intensity accompanied by a discernible red shift in the emission maxima [27] . We have also examined the aggregation propensity of Trypsin (50 μM) in presence of 50 % EtOH (Figure S1b), which is the maximum alcohol (EtOH) content in most of the alcoholic beverages, [13] and we did not observe the formation of Trypsin fibrils (Trypsin Fib).…”

“…[26] In the native state, the tryptophan amino acid residue(s) experience a hydrophobic environment, and hence its inherent fluorescence remains quenched, a condition referred to as "native quenching". [27] However, in the presence of 60 % EtOH, Trypsin gets denatured and the active intrinsic fluorophores experience a more hydrophilic environment and the native quenching is lost. This results in the rise in fluorescence intensity accompanied by a discernible red shift in the emission maxima.…”

Insights into cyanobacterial alkane biosynthesis

“…However, some reports suggest that ADO is a very slow enzyme with the highest turnover reaches to ∼1 min –1 (Andre et al, 2013 ). Also, AAR protein is prone to aggregation when overexpressed in E. coli , leading to poor in vivo and in vitro catalytic activities (Kudo et al, 2019 ; Sharma et al, 2021 ). ADO from N. punctiforme and AAR from S. elongatus PCC7942 were found to be most efficient amongst various homologues tested (Kudo et al, 2016 ; Zhu et al, 2018 ).…”

“…One of the strategies to enhance alkane production is to engineer the AAR enzyme to increase its activity as it acts as a rate-limiting enzyme. Our group has recently shown the propensity of AAR to aggregate when overexpressed in E. coli (Sharma et al, 2021 ), suggesting the need for an improved AAR enzyme. In comparison with other model organisms such as E. coli and S. cerevisiae , limited genetic tools are accessible for manipulating the cyanobacterial genome.…”