Stenotrophomonas maltophilia OleC‐Catalyzed ATP‐Dependent Formation of Long‐Chain Z‐Olefins from 2‐Alkyl‐3‐hydroxyalkanoic Acids

Abstract

The bacterial pathway of olefin biosynthesis starts with OleA catalyzed "head-to-head" condensation of two CoA-activated long-chain fatty acids to generate (R)-2-alkyl-3-ketoalkanoic acids. A subsequent OleD-catalyzed reduction generates (2R,3S)-2-alkyl-3-hydroxyalkanoic acids. We now show that the final step in the pathway is an OleC-catalyzed ATP-dependent decarboxylative dehydration to form the corresponding Z olefins. Higher kcat /Km values were seen for substrates with longer alkyl chains. All four stereoisomers of 2-hexyl-3-hydroxydecanoic acid were shown to be substrates, and GC-MS and NMR analyses confirmed that the product in each case was (Z)-pentadec-7-ene. LC-MS analysis supported the formation of AMP adduct as an intermediate. The enzymatic and stereochemical course of olefin biosynthesis from long-chain fatty acids by OleA, OleD and OleC is now established.

β-Lactone Synthetase Found in the Olefin Biosynthesis Pathway

“…We believe that thermal decomposition during GC/MS analysis caused the product of OleC catalysis to be misidentified. Additionally, when reviewing supplemental NMR data from the literature report of Sm OleC characterization, resonances of the cis- and trans -β-lactones, consistent with those synthesized here, are visible 12 .…”

Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis

“…In this study, all four X. campestris Ole proteins were purified in a recombinant form from separate E. coli expression host cell lines. In contrast to conclusions reached in a previous study (11), we found that OleB is required for the reconstitution of hydrocarbon biosynthesis, consistent with the ubiquitous occurrence of oleB in the olefin gene cluster. The OleA and OleD reactions were previously demonstrated to produce a ␤-keto acid and ␤-hydroxy acid, respectively ( Fig.…”

“…The purification of individually expressed OleA, OleC, and OleD proteins derived from different bacteria had been reported previously (5,(7)(8)(9)(10)(11), while the purification of an OleB protein is reported here for the first time, to our knowledge. In this study, all four X. campestris Ole proteins were purified in a recombinant form from separate E. coli expression host cell lines.…”

“…1) (7,9). OleC, purified from Stenotrophomonas maltophilia, was reported to produce an olefin (11), but we recently demonstrated that OleC from that organism produces a ␤-lactone and that OleB is required to transform that to the final olefinic hydrocarbon (10).…”

“…However, OleC is now known to react with the product of the OleD reaction to generate a thermally labile ␤-lactone (10). The ␤-lactone undergoes spontaneous and nonbiological decarboxylation to an olefin when monitored by gas chromatography (GC), which led to the initial incorrect assignment of OleC function (10,11). Recent data suggest that OleB acts as an unprecedented ␤-lactone decarboxylase to yield the final olefin product (10).…”