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 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.IMPORTANCE Bacteria 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 in X. campestris The 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.

Keywords: bacteria; hydrocarbon; multienzyme complex; olefin.

FIG 1

Olefin biosynthesis enzymatic pathway and gene clusters. Gene clusters 1 and 2 are the two most common gene arrangements found in oleABCD-containing organisms. The two fatty acyl-CoAs are typically linear alkyl chains between 10 and 16 carbons in length.

FIG 2

Plasmid expression, purification, and SDS-PAGE analysis of Ole protein coexpression. (A) The table represents all tag combinations constructed. The placement of the His₆ and FLAG tags on the left and right of the protein corresponds to N- and C-terminal tags, respectively. Colors in the table represent the grouping of genes on a single vector for expression. (B) Purification of OleBCD (tag arrangement 4) over His affinity column with increasing amounts of imidazole. Peak fractions indicated by the bar were collected, concentrated, and loaded onto the anti-FLAG column (lane labeled “Load” on gel in panel C). (C) SDS-PAGE showing the post-His affinity column concentrate and the anti-FLAG purification of OleBCD tag arrangement 4. Bound samples were washed twice (W1 and W2) with buffer before elution with FLAG peptide. Note that OleC, the weakest-intensity band, contained the FLAG tag. Abs, absorbance.

FIG 3

Anti-OleC immunoblot on a nondenaturing protein gel. Native OleC from the supernatant of lysed X. campestris cells migrates closely with recombinantly expressed and purified X. campestris OleBCD rather than individually purified OleC. Xc, X. campestris.

FIG 4

Size exclusion chromatography of Ni²⁺ column-purified OleBCD (tag arrangement 4). The peak absorbance at 65.0 ml corresponded to an estimated size of 1.9 MDa when extrapolated from protein standards. Two hundred microliters of each 4-ml fraction was tested for OleBCD activity by the addition of OleA, myristoyl-CoA, and cofactors.

FIG 5

Electron micrograph of OleBCD assemblies with particle size analysis. (A) OleBCD assemblies by TEM. (B) Histogram analysis of >150 particles from four different frames, with representative micrographs shown below. Measurement was of the greatest particle diameter. Average assembly size is 30.3 nm, and the median is 29.6 nm.

FIG 6

Model for the biosynthesis of long-chain head-to-head olefins in X. campestris showing that OleA acts predominantly as a soluble cytosolic dimer, condensing acyl-CoAs, docking, and providing its reaction product to the OleBCD complex, which ultimately releases a long-chain olefin.