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 from Xanthomonas campestris in Escherichia coli and purification over His6 and 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 with X. campestris crude 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.
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We thank Isaac Hamilton for his help with gas chromatography assays and standard curves, as well as Iwaki Shigehiro for his help in the purification of Ole proteins. We also thank the University of Minnesota Center for Mass Spectrometry and Proteomics for their help in peptide fragment identification. J.K.C. was supported in part by NIH Training for Future Biotechnology Development (grant T32GM008347). M.R.J. was supported in part by an NIH Chemistry-Biology Interface training grant (T32GM008700). We acknowledge the support of the Biotechnology Institute from the University of Minnesota and the MnDRIVE Initiative from the Office of the Vice President for Research of the University of Minnesota (to C.M.W. and L.P.W.). The electron micrographs were collected using a Tecnai TF30 TEM maintained by the Characterization Facility, College of Science and Engineering, University of Minnesota.
- Multienzyme complex