Cyanobacterial alkane biosynthesis is catalyzed by acyl-(acyl carrier protein (ACP)) reductase (AAR) and aldehyde-deformylating oxygenase (ADO) in a two-step reaction. AAR reduces acyl-ACPs to fatty aldehydes, which are then converted by ADO to alkanes, the main components of diesel fuel. Interaction between AAR and ADO allows AAR to efficiently deliver the aldehyde to ADO. However, this interaction is poorly understood. Here, using analytical size-exclusion chromatography (SEC), we show that electrostatic interactions play an important role in the binding of the two enzymes. Alanine-scanning mutagenesis at charged residues around the substrate entry site of ADO revealed that E201A mutation greatly reduced hydrocarbon production. SEC measurement of the mutant demonstrated that E201 of ADO is essential for the AAR–ADO interaction. Our results suggest that AAR binds to the substrate entrance gate of ADO and thereby facilitates the insertion of the reactive and relatively insoluble aldehyde into the hydrophobic channel of ADO. Abbreviations: AAR: acyl-ACP reductase; ACP: acyl carrier protein; ADO: aldehyde-deformylating oxygenase; ASA: solvent accessible surface area; BSA: bovine serum albumin; CD: circular dichroism; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; GC-MS: gas chromatography-mass spectrometer; HPLC: high-performance liquid chromatography; IPTG: isopropyl-β-D-thiogalactoside; MRE: mean residue ellipticity; NpAAR: AAR from Nostoc punctiforme PCC 73102; NpADO: ADO from Nostoc punctiforme PCC 73102; PmADO: ADO from Prochlorococcus marinus MIT 9313; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SeAAR: AAR from Synechococcus elongatus PCC 7942; SeADO: ADO from Synechococcus elongatus PCC 7942; SEC: size-exclusion chromatography; TeAAR: AAR from Thermosynechococcus elongatus BP-1; TeADO: ADO from Thermosynechococcus elongatus BP-1; UV: ultraviolet

Hen eggs are rich in proteins and are an important source of protein for humans. Pasteurized frozen whole hen eggs are widely used in cooking and confectionery and can be stored for long periods. However, processed eggs differ from raw eggs in properties such as viscosity, foaming ability, and thermal aggregation. To develop pasteurized frozen whole egg products with properties similar to those of unpasteurized whole eggs, it is necessary to establish a method that can differentiate between the two egg types with respect to the structures of their proteins. In this study, size-exclusion chromatography (SEC) and SEC coupled with small-angle X-ray scattering (SEC-SAXS) were successfully used to differentiate between the proteins in unpasteurized and pasteurized frozen whole eggs. We found that proteins in the plasma fraction of egg yolk, especially apovitellenins I and II, formed large aggregates in the pasteurized eggs, indicating that their structures are sensitive to temperature changes during pasteurization, freezing, and thawing. The results suggest that SEC and SEC-SAXS can be used to differentiate between unpasteurized and pasteurized frozen whole eggs. Additionally, they may be useful in determining molecular sizes and shapes of multiple components in various complex biological systems such as whole eggs.