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

  • Janice A. Frias
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Jack E. Richman
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Jasmine S. Erickson
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Lawrence P. Wackett
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology, and Biophysics, 140 Gortner Laboratory, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108. Tel.: 612-625-3785; Fax: 612-624-5780
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Author Footnotes
    * This work was supported by a University of Minnesota doctoral dissertation fellowship (to J. A. F.) and by Department of Energy ARPA-E Award DE-AR0000007 and the Initiative for Renewable Energy and the Environment (to L. P. W.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Method S1 and Figs. S1 and S2.
Open AccessPublished:January 25, 2011DOI:https://doi.org/10.1074/jbc.M110.216127
      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 C8 to C16 in length. With limiting myristoyl-CoA (C14), 1 mol of the free coenzyme A was released/mol of myristoyl-CoA consumed. Using [14C]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.

      Introduction

      Commodity hydrocarbons derive from petroleum, but nature provides a rich source of hydrocarbons for which biosynthetic pathways are being elucidated. Isoprenoid biosynthesis has been well studied (
      • Muntendam R.
      • Melillo E.
      • Ryden A.
      • Kayser O.
      ), and an enzymatic decarbonylation of fatty aldehydes to produce alkanes has recently been demonstrated for cyanobacteria (
      • Schirmer A.
      • Rude M.A.
      • Li X.
      • Popova E.
      • del Cardayre S.B.
      ). It has been known for more than 40 years that some bacteria biosynthesize long (C23–C33) hydrocarbon chains containing a double bond at the median carbon via a mechanism known as a “head-to-head” condensation of fatty acyl groups (
      • Tornabene T.G.
      • Oró J.
      ,
      • Albro P.W.
      • Dittmer J.C.
      ,
      • Albro P.W.
      • Dittmer J.C.
      ,
      • Albro P.W.
      • Dittmer J.C.
      ,
      • Albro P.W.
      • Dittmer J.C.
      ,
      • Albro P.W.
      • Meehan T.D.
      • Dittmer J.C.
      ). For example, bacteria from the genus Arthrobacter produce largely C15 fatty acids (
      • Unell M.
      • Kabelitz N.
      • Jansson J.K.
      • Heipieper H.J.
      ) and make predominantly C29 olefins (
      • Frias J.A.
      • Richman J.E.
      • Wackett L.P.
      ). These observations are consistent with studies in 1969 showing the loss of the 14C label at carbon-1 of one of the acyl groups undergoing head-to-head condensation (
      • Albro P.W.
      • Dittmer J.C.
      ). These early in vitro studies were conducted with crude cell protein extracts. It was not until 2010 that the genes involved in the head-to-head biosynthetic pathway were described in the peer-reviewed literature (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ,
      • Sukovich D.J.
      • Seffernick J.L.
      • Richman J.E.
      • Hunt K.A.
      • Gralnick J.A.
      • Wackett L.P.
      ), providing new insights into the biosynthetic pathway based on a bioinformatics analysis of the gene and protein families.
      OleA is homologous to proteins in the thiolase or condensing enzyme superfamily (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ,
      • Sukovich D.J.
      • Seffernick J.L.
      • Richman J.E.
      • Gralnick J.A.
      • Wackett L.P.
      ). This is a very large superfamily of over 13,000 known proteins. The known thiolase superfamily proteins typically catalyze condensation reactions between acyl-thioester substrates, either with or without the loss of a carboxyl group. Approximately 70 bacteria are known to contain genes denoted as oleABCD, and those tested produce long-chain olefinic hydrocarbons (
      • Sukovich D.J.
      • Seffernick J.L.
      • Richman J.E.
      • Gralnick J.A.
      • Wackett L.P.
      ). The precise role of each ole gene product in the biosynthesis remains to be defined. When the oleC gene is deleted or only the oleA gene is present in vivo, a long-chain ketone(s) is observed. These data supported the idea that OleA is involved in the initial stages of the head-to-head hydrocarbon biosynthetic reactions (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ,
      • Sukovich D.J.
      • Seffernick J.L.
      • Richman J.E.
      • Gralnick J.A.
      • Wackett L.P.
      ,

      Friedman, L., Rude, M., (September 18, 2008) International Patent WO2008/113041.

      ).
      There are two alternative proposals in the literature regarding the OleA condensation reaction (Fig. 1). Beller et al. (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ) (Fig. 1A) have proposed that OleA catalyzes a decarboxylative condensation between a β-ketoacyl-CoA and a fatty acyl-CoA. Sukovich et al. (
      • Sukovich D.J.
      • Seffernick J.L.
      • Richman J.E.
      • Gralnick J.A.
      • Wackett L.P.
      ) (Fig. 1B) have proposed that OleA catalyzes a non-decarboxylative Claisen condensation between two fatty acyl-CoA substrates. These two types of condensation reactions are difficult to differentiate in vivo, where both fatty acyl-CoAs and β-ketoacyl-CoAs may be present simultaneously, and many enzymes are present. The study by Beller et al. (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ) used a purified OleA enzyme, but their demonstration of activity required the addition of a crude soluble protein extract from Escherichia coli. The proposed β-ketoacyl-CoA substrate was suggested to have been generated from the corresponding acyl-CoA by the proteins present in the E. coli soluble fraction. A clear differentiation between OleA reaction A and B could be obtained using a purified OleA preparation in admixture with defined substrates in vitro. The two types of condensation reactions could also be differentiated by determining the reaction product. OleA reaction A produces a 1,3-diketone, whereas OleA reaction B yields a β-ketoacid (Fig. 1).
      Figure thumbnail gr1
      FIGURE 1Fundamentally different condensation mechanisms have been proposed for OleA: decarboxylative condensation between a β-keto ester and an acyl thioester (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ) (A) or non-decarboxylative condensation between two acyl thioesters (
      • Sukovich D.J.
      • Seffernick J.L.
      • Richman J.E.
      • Gralnick J.A.
      • Wackett L.P.
      ) (B).
      There are other important questions that can be answered directly using a purified OleA protein and purified single substrates. These include determining the substrate specificity of OleA with respect to chain length, determining the complete reaction stoichiometry, determining what drives the apparent Claisen condensation to completion, and revealing why cloning oleA genes in heterologous hosts produces monoketones. These issues are addressed in the present work.
      The OleA protein from Xanthomonas campestris was cloned, overexpressed in E. coli, and purified to homogeneity. The putative product of the reaction was synthesized chemically to allow comparison with the biochemical product. OleA was shown to react with myristoyl
      Myristoyl is equivalent to tetradecanoyl.
      -CoA to produce the corresponding β-ketoacid via a non-decarboxylative Claisen condensation reaction. This intermediate was shown to react, in the presence of OleC and OleD, to yield a long-chain olefin. In the absence of OleC and OleD, the product of the OleA reaction was shown to undergo spontaneous chemical decarboxylation to yield a ketone. This explains previous in vivo observations of ketone formation with the expression of an oleA gene in a heterologous host (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ,
      • Sukovich D.J.
      • Seffernick J.L.
      • Richman J.E.
      • Gralnick J.A.
      • Wackett L.P.
      ).

      DISCUSSION

      In this study, the OleA protein from X. campestris was purified to homogeneity and shown to condense fatty acyl-CoA substrates to produce a condensed β-ketoacid with the release of 2 mol of CoA. The β-ketoacid, synthesized chemically or enzymatically, was shown to undergo further metabolism to yield a long-chain olefin in the presence of OleC and OleD. These studies confirmed that OleA catalyzes the first reaction in alkene biosynthesis with acyl-CoA substrates and carries out a non-decarboxylative Claisen condensation reaction.
      An OleA protein was previously purified from M. luteus, and it was proposed to catalyze a different reaction (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ) than the one demonstrated here with the OleA protein from X. campestris. The Xanthomonas and Micrococcus OleA proteins showed 38% sequence identity (Table 1) in a pairwise alignment of their amino acid sequences (
      • Altschul S.F.
      • Madden T.L.
      • Schäffer A.A.
      • Zhang J.
      • Zhang Z.
      • Miller W.
      • Lipman D.J.
      ), so they could conceivably catalyze different reactions. The oleA genes from both organisms cluster with oleBCD genes. In the Micrococcus genome, the oleB and oleC genes are fused and likely produce a multidomain protein. However, the OleA, OleB, OleC, and OleD domains are present in both organisms. It was shown in the present study that OleC and OleD proteins act on the β-ketoacid product generated by X. campestris OleA to produce a long-chain olefin. When the Micrococcus oleA gene was cloned and expressed in E. coli, long-chain ketones were observed (
      • Beller H.R.
      • Goh E.B.
      • Keasling J.D.
      ). In the present study, the recombinant E. coli strain expressing the X. campestris OleA protein alone was also observed to produce long-chain ketones that were not observed in the wild-type E. coli (data not shown). The in vitro data in this study showed that the ketones readily arise from the decarboxylation of a corresponding β-ketoacid intermediate. These observations are all consistent with a non-decarboxylative Claisen condensation as shown in Fig. 1B and difficult to reconcile with the proposed decarboxylative reaction shown in Fig. 1A.
      The reaction catalyzed by OleA is somewhat reminiscent of the Zoogloea thiolase reaction that catalyzes the first step in the biosynthesis of polyhydroxybutyrate (
      • Davis J.T.
      • Moore R.N.
      • Imperiali B.
      • Pratt A.J.
      • Kobayashi K.
      • Masamune S.
      • Sinskey A.J.
      • Walsh C.T.
      • Fukui T.
      • Tomita K.
      ). In the latter reaction, however, the condensed product is a β-ketoacetyl-CoA, acetoacetyl-CoA, and with OleA, the product is a β-keto acid. Several lines of evidence strongly suggested that OleA does not produce a β-ketoacetyl-CoA that is hydrolyzed to the acid by another enzyme. First, the oleA gene was cloned as a single open reading frame (ORF) from synthetic DNA and expressed in E. coli, a bacterium that does not natively synthesize hydrocarbons. Enzymes capable of hydrolyzing 2-myristoylmyristoyl-CoA are not likely to be present in E. coli. Second, OleA was highly purified as shown by SDS-PAGE (Fig. 2), so even the unlikely E. coli hydrolytic enzyme would have been removed. Last, our HPLC conditions would have detected 2-myristoylmyristoyl-CoA, and this was never detected.
      Based on the data obtained and the known role of the conserved cysteine found in other members of the thiolase superfamily, a working reaction mechanism can be presented for the OleA-catalyzed reaction (Fig. 7). We propose that initially an active site cysteine in the resting enzyme (Fig. 7A) is acylated, and coenzyme A is liberated (Fig. 7B). Subsequently, the tethered substrate is probably activated by an active site base to yield a carbanion on the tethered substrate (Fig. 7C). The carbanion then can react at the active site with the carbonyl carbon of a non-covalently bound acyl-CoA (Fig. 7D). That reaction forms a carbon-to-carbon bond with the condensed product still tethered to the enzyme cysteine and producing the second molecule of coenzyme A formed in the reaction cycle (Fig. 7E). The covalently bound condensation product can then undergo hydrolysis to yield the final β-ketoacid product and regenerate the free cysteine residue of the resting enzyme state (Fig. 7A). Although several features of this proposed mechanism are not yet demonstrated directly, there are multiple data that support this proposal. First, this mechanism explains the observed stoichiometry in which 2 mol of coenzyme A are observed/mol of condensed product. Second, the observed high rate of hydrolysis of acyl-CoAs to produce fatty acids is not unexpected if the enzyme has a mechanism to hydrolyze thioester-linked intermediates during its normal reaction cycle. Thus, there could be a kinetic competition between hydrolysis of the initially bound acyl group (Fig. 7B) and the tethered condensation product (Fig. 7E). Depending upon the binding affinity for the different length acyl-CoA used in the experiment described in Table 3, hydrolysis of intermediate 7B or 7E would occur preferentially. Last, proteins in the thiolase superfamily typically use an active site cysteine to acquire an acyl chain to initiate catalysis (
      • Heath R.J.
      • Rock C.O.
      ,
      • Haapalainen A.M.
      • Meriläinen G.
      • Wierenga R.K.
      ), and the region around the cysteine residue shown in Table 1 is the most highly conserved region of OleA with other members of the superfamily.
      Figure thumbnail gr7
      FIGURE 7Proposed reaction cycle for OleA. The top of the cycle (A) shows the resting enzyme that reacts with an acyl-CoA to start the reaction cycle; B, enzyme with the covalently attached acyl chain; C, enzyme having abstracted a proton from the acyl chain to activate it; D, activated acyl chain reacting with the second acyl chain bound to the enzyme; E, condensed fatty acyl chains still bonded to the enzyme system just prior to hydrolytic release. CoASC(O)CH2R1 and CoASC(O)CH2R2, first and second reacting acyl-coenzyme A, respectively. B: attached by a line to Enz represents an enzyme base. The products of the reaction, two molecules of coenzyme A (CoASH) and a β-keto acid, are highlighted by boxes.
      There are significant questions that remain to be addressed regarding this proposed mechanism (Fig. 7). First, the identity of the proposed cysteine nucleophile has not been directly demonstrated here. Second, the suggested generation of a carbanion (Fig. 7B) requires a general base that remains to be identified. Additionally, this mechanism would be supported by the identification of the binding sites for the acyl chains and that the chains are covalently and non-covalently bound, respectively.
      This study identified the product of the OleA-catalyzed reaction to be a β-keto acid. The production of olefins required the presence of OleC and OleD in addition to OleA. These data indicated that OleC and OleD catalyze further reactions with the β-ketoacid intermediate generated by OleA. This was supported by experiments in which 2-myristoylmyristic acid was transformed to an olefin by OleC and OleD. The corresponding ketone was not transformed to an olefin, consistent with the idea that the ketone is not a physiologically relevant intermediate. There is also the issue that C-2 in 2-myristoylmyristic acid is a chiral center. The synthetic 2-myristoylmyristic acid is racemic, and it is plausible that only one enantiomer will react with OleD. The chirality of the reaction is currently under investigation.

      Acknowledgments

      We thank David Sukovich for providing a selection of oleA genes used in these studies. We acknowledge Fred Schendel and Mary Pruss for fermentation and cell harvesting. We especially thank Drs. Sharon Murphy and Linda von Weymarn for assistance with the HPLC radioflow equipment. We thank Brandon Goblirsch and Carrie Wilmot for insightful discussions regarding the OleA mechanism described here. We thank Dr. Burckhard Seelig and laboratory members J. Haugen and L. Hagman for the use of and for instruction in the use of the FPLC and size exclusion column. Mass spectrometry (ESI only) was conducted in the Center for Mass Spectrometry and Proteomics and Mass Spectrometry Services in the Masonic Cancer Center with the assistance of Tom Krick, Brock Matter, and Peter Villalta.

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