title: Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis.
abstract: Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins fromXanthomonas campestrisinEscherichia coliand purification over His6and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted withX. campestriscrude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction.IMPORTANCEBacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that long-chain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies inX. campestrisThe OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota, USA.|BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: James K
authors.initials: JK
authors.lastname: Christenson
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota, USA.|BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Jensen
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Brandon R
authors.initials: BR
authors.lastname: Goblirsch
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Fatuma
authors.initials: F
authors.lastname: Mohamed
authors.affiliation: Department of Diagnostic and Biological Sciences, University of Minnesota, Minneapolis, Minnesota, USA.|Characterization Facility, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota, USA.|BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Carrie M
authors.initials: CM
authors.lastname: Wilmot
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota, USA wacke003@umn.edu.|BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.|Microbial and Plant Genomic Institute, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: Wackett
mesh_terms: D000475:Alkenes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D053898:Biosynthetic Pathways; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D006838:Hydrocarbons / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D016959:Xanthomonas campestris / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000475:Alkenes; D001426:Bacterial Proteins; D006838:Hydrocarbons; D009097:Multienzyme Complexes
keywords: bacteria; hydrocarbon; multienzyme complex; olefin