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 His 6 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 ␤-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 longchain 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.

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

“…It is hypothesized that the final decarboxylation to form the cis -olefin product is catalyzed by OleB (α/β-hydrolase superfamily) [10]. OleBCD were proposed to form a multi-enzyme assembly that may function to efficiently move the hydrophobic pathway intermediates between enzyme active sites, preserve the stereochemistry, and sequester the highly reactive β-lactone from the cell [11]. As well as initiating the pathway, OleA has been proposed to act as a shuttle between fatty acid metabolism and the OleBCD assembly feeding its β-keto acid product to OleD [11].…”

Distribution and diversity of olefins and olefin-biosynthesis genes in Gram-positive bacteria

“…The lactone synthase OleC forms a heat-labile internal ester between the β-hydroxy and the carboxyl group and one molecule of water is released. Finally, the lactone decarboxylase OleB liberates one molecule of CO 2 and converts the internal lactone into an alkene with a cis-configured internal double bond (internal olefins) at the connection point of the two original fatty acid precursors (Beller et al [3]) (Sukovich et al [14]) (Frias et al [7]) (Christenson et al [4]) (Christenson, Richman et al [6]) (Christenson, Jensen et al [5]).…”

“…The nature of such internal olefin hydrocarbons was first characterized in the Gram-positive Sarcina lutea (now Kocuria rhizophila) (Albro and Dittmer [1]) (Tornabene et al [17]), and the oleABCD genes were first described in the Gram-positive Micrococcus luteus NCTC2665 (Beller et al [3]). Nevertheless, the catalytic mechanism of OleA as well as the order and nature of the reaction steps of the OleABCD enzymes, as summarized above, were finally elucidated mainly in the Gram-negative species Shewanella oneidensis (Sukovich et al [14]) and Xanthomonas campestris (Frias et al [7]) (Christenson et al [4]) (Christenson et al [6]) (Christenson et al [5]). In addition, the first report on the phylogenetic distribution and diversity of olefin production in bacteria by Sukovich et al [13] described bacterial olefin production as a predominately Gram-negative capability using species from four Gram-negative phyla (Proteobacteria, Planctomycetes, Chloroflexi, Verrucomicrobia) and one Gram-positive phylum (Actinobacteria) (Sukovich et al [13]).…”

Linkage of Marine Bacterial Polyunsaturated Fatty Acid and Long-Chain Hydrocarbon Biosynthesis

“…OleC, a β-lactone synthetase, then generates a β-lactone moiety (Christenson et al, 2017b) which is subsequently de-carboxylated by OleB, a β-lactone decarboxylase, yielding the final olefin product (Christenson et al, 2017c). Recent work has demonstrated that OleBCD together form a large multimeric enzyme complex that processes the β-keto intermediate formed by OleA activity (Christenson et al, 2017a). Weak interactions between the OleBCD complex and OleA in vitro suggests that OleA condenses the precursor acyl groups and possibly transfers the β-keto acid product directly to the OleBCD complex for further processing (Christenson et al, 2017a).…”

“…Recent work has demonstrated that OleBCD together form a large multimeric enzyme complex that processes the β-keto intermediate formed by OleA activity (Christenson et al, 2017a). Weak interactions between the OleBCD complex and OleA in vitro suggests that OleA condenses the precursor acyl groups and possibly transfers the β-keto acid product directly to the OleBCD complex for further processing (Christenson et al, 2017a). In the absence of downstream processing by OleBCD, the OleA catalyzed β-keto acid intermediate undergoes spontaneous decarboxylation to form a ketone product (Sukovich et al, 2010b; Frias et al, 2011; Figure 1).…”