Electrostatic interactions at the interface of two enzymes are essential for two-step alkane biosynthesis in cyanobacteria

Abstract

ABSTRACT 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