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.

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In their Letter to the Editor, Beller et al. (1) suggest a reinterpretation of the mass spectrum in Fig. 6A of our paper (2). The full mass spectrum is shown here in the top panel of Fig. 1. They make the point that long-chain alkenes show weak molecular ions, e.g. 3-15% and high abundance ions in the m/z ϳ40 -120 region. That's exactly what we saw. We showed only the high molecular weight region because we obtained a relatively strong molecular ion peak and had already identified the same alkene in a different set of experiments that were presented in Fig. 5. The mass spectrum for that alkene is shown in the middle panel of Fig. 1. Beller et al. (1) contend that our compound is dramatically different from heptacosene based on a mass spectrum presented in a patent application (3) that represented heptacosene. We show here in Fig. 1 that mass spectrum from Ref. 3 (bottom panel) and the mass spectra of our compounds that were characterized in Figs. 5 and 6 of our paper (2). All of the major peaks and nested mass fragments are virtually identical. The molecular ions are present in all at m/z 378 and in the range of 3-15% relative abundance. This recapitulates the point we made in Figs. 5 and 6 of the paper. Incubating OleA, OleC, OleD, and myristoyl-CoA yielded 14-heptacosene. Incubating myristoylmyristic acid, OleC, and OleD yielded 14-heptacosene. This supported the other data in the paper showing that OleA condenses acyl-CoA substrates to make ␤-keto acid products via a non-decarboxylative Claisen condensation reaction.