Purification and Characterization of OleA from Xanthomonas campestris and Demonstration of a Non-decarboxylative Claisen Condensation Reaction

Journal Article · Fri Mar 25 04:00:00 EDT 2011 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M110.216127· OSTI ID:1211356

Frias, JA; Richman, JE; Erickson, JS; Wackett, LP

OleA catalyzes the condensation of fatty acyl groups in the first step of bacterial long-chain olefin biosynthesis, but the mechanism of the condensation reaction is controversial. In this study, OleA from Xanthomonas campestris was expressed in Escherichia coli and purified to homogeneity. The purified protein was shown to be active with fatty acyl-CoA substrates that ranged from C(8) to C(16) in length. With limiting myristoyl-CoA (C(14)), 1 mol of the free coenzyme A was released/mol of myristoyl-CoA consumed. Using [(14)C] myristoyl-CoA, the other products were identified as myristic acid, 2-myristoylmyristic acid, and 14-heptacosanone. 2-Myristoylmyristic acid was indicated to be the physiologically relevant product of OleA in several ways. First, 2-myristoylmyristic acid was the major condensed product in short incubations, but over time, it decreased with the concomitant increase of 14-heptacosanone. Second, synthetic 2-myristoylmyristic acid showed similar decarboxylation kinetics in the absence of OleA. Third, 2-myristoylmyristic acid was shown to be reactive with purified OleC and OleD to generate the olefin 14-heptacosene, a product seen in previous in vivo studies. The decarboxylation product, 14-heptacosanone, did not react with OleC and OleD to produce any demonstrable product. Substantial hydrolysis of fatty acyl-CoA substrates to the corresponding fatty acids was observed, but it is currently unclear if this occurs in vivo. In total, these data are consistent with OleA catalyzing a non-decarboxylative Claisen condensation reaction in the first step of the olefin biosynthetic pathway previously found to be present in at least 70 different bacterial strains.

• Sponsoring Organization: USDOE Advanced Research Projects Agency - Energy (ARPA-E)
• DOE Contract Number: AR0000007
• OSTI ID: 1211356
• Journal Information: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 13 Vol. 286; ISSN 0021-9258
• Country of Publication: United States
• Language: English