Purification and characterization of OleA from Xanthomonas campestris and demonstration of a non-decarboxylative claisen condensation reaction

Article abstract of DOI: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 C ₈ to C₁₆ in length. With limiting myristoyl-CoA (C ₁₄), 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 reactionin the first step of the olefin biosynthetic pathway previously found to be presentin at least 70 different bacterial strains.

Full text of DOI:10.1074/jbc.M110.216127

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 13, pp. 10930–10938, April 1, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Purification and Characterization of OleA from Xanthomonas
campestris and Demonstration of a Non-decarboxylative
□
S
Claisen Condensation Reaction*
Received for publication, December 23, 2010, and in revised form, January 24, 2011 Published, JBC Papers in Press, January 25, 2011, DOI 10.1074/jbc.M110.216127
Janice A. Frias, Jack E. Richman, Jasmine S. Erickson, and Lawrence P. Wackett1
From the Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota,
St. Paul, Minnesota 55108
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 pro-
tein 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 myris-
toyl-CoA consumed. Using [14C]myristoyl-CoA, the other prod-
ucts 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, syn-
thetic 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 stud-
ies. The decarboxylation product, 14-heptacosanone, did not react
with OleC and OleD to produce any demonstrable product. Sub-
stantial hydrolysis of fatty acyl-CoA substrates to the correspond-
ing 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-decarboxylativeClaisencondensationreactioninthefirststep
oftheolefinbiosyntheticpathwaypreviouslyfoundtobepresentin
at least 70 different bacterial strains.
hydrocarbon chains containing a double bond at the median
carbon via a mechanism known as a “head-to-head” condensa-
tion of fatty acyl groups (3–8). For example, bacteria from the
genus Arthrobacter produce largely C15 fatty acids (9) and make
predominantly C29 olefins (10). These observations are consis-
tent 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 (6). 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 (11, 12), providing
new insights into the biosynthetic pathway based on a bioinfor-
matics analysis of the gene and protein families.
OleA is homologous to proteins in the thiolase or condensing
enzyme superfamily (11, 13). 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 con-
tain genes denoted as oleABCD, and those tested produce long-
chain olefinic hydrocarbons (13). 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 (11, 13, 14).
There are two alternative proposals in the literature regard-
ing the OleA condensation reaction (Fig. 1). Beller et al. (11)
(Fig. 1A) have proposed that OleA catalyzes a decarboxylative
condensation between a ␤-ketoacyl-CoA and a fatty acyl-CoA.
Sukovich et al. (13) (Fig. 1B) have proposed that OleA catalyzes
a non-decarboxylative Claisen condensation between two fatty
acyl-CoA substrates. These two types of condensation reac-
tions are difficult to differentiate in vivo, where both fatty acyl-
CoAs and ␤-ketoacyl-CoAs may be present simultaneously,
and many enzymes are present. The study by Beller et al. (11)
used a purified OleA enzyme, but their demonstration of activ-
ity required the addition of a crude soluble protein extract from
Escherichia coli. The proposed ␤-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 reac-
tions could also be differentiated by determining the reaction
Commodity hydrocarbons derive from petroleum, but
nature provides a rich source of hydrocarbons for which bio-
synthetic pathways are being elucidated. Isoprenoid biosynthe-
sis has been well studied (1), and an enzymatic decarbonylation
of fatty aldehydes to produce alkanes has recently been demon-
strated for cyanobacteria (2). It has been known for more than
40 years that some bacteria biosynthesize long (C23–C33)
* 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 Environ-
ment (to L. P. W.).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Method S1 and Figs. S1 and S2.
1 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; E-mail: wacke003@umn.edu.
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Purification and Characterization of OleA
Carefully monitored hydrogenolysis of benzyl 2-myris-
toylmyristate (Pd/C catalyst, methyl t-butyl ether solvent), iso-
lated by filtration and cooling of the filtrate to Ϫ80 °C, pro-
duced a 5:2 (mol/mol) mixture of 2-myristoylmyristic acid and
its decarboxylated product 14-heptacosanone as determined by
GC-MS analysis after methylation of the acid (CH2N2). On cold
storage (Ϫ80 °C), the solution was enriched to 8:1 2-myris-
toylmyristic acid and the derived ketone, apparently by prefer-
ential precipitation of the ketone.
Cloning and Expression of OleA—Synthetic oleA genes were
designed based on oleA genes from Congregibacter litoralis
KT71 (ZP_01103251.1), Xanthomonas campestris spv. campes-
tris str. ATCC 33913 (NP_635607.1), Xylella fastidiosa 9a5c
(NP_299252.1), Plesiocystis pacifica SIR-1 (ZP_01906524.1),
and ␥-proteobacterium NOR5-3 (ZP_05127044.1) (see supple-
mental Fig. S1) and purchased from DNA 2.0 (Menlo Park, CA).
The genes were cut with NdeI and BamHI restriction enzymes
and cloned into pET28bϩ (Novagen, Madison, WI). All five
genes were separately transformed into E. coli One Shot BL21
(DE3) (Invitrogen). All five recombinant strains were screened
for soluble protein expression in 50-ml cultures induced for 4 h
at 37 °C. Two of the five constructs expressed soluble protein in
E. coli; only X. campestris was found to be active in vitro, and
that was selected for further study.
FIGURE1. Fundamentallydifferentcondensationmechanismshavebeen
proposed for OleA: decarboxylative condensation between a ␤-keto
ester and an acyl thioester (11) (A) or non-decarboxylative condensation
between two acyl thioesters (13) (B).
product. OleA reaction A produces a 1,3-diketone, whereas
OleA reaction B yields a ␤-ketoacid (Fig. 1).
There are other important questions that can be answered
directly using a purified OleA protein and purified single sub-
strates. 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 chemi-
cally to allow comparison with the biochemical product. OleA
was shown to react with myristoyl2-CoA to produce the corre-
sponding ␤-ketoacid via a non-decarboxylative Claisen con-
densation 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 reac-
tion was shown to undergo spontaneous chemical decarboxy-
lation to yield a ketone. This explains previous in vivo observa-
tions of ketone formation with the expression of an oleA gene in
a heterologous host (11, 13).
E. coli for OleA purification was cultivated under two differ-
ent conditions. Small scale cultivations were conducted in 2-li-
ter flasks containing 500 ml of LB with 50 ␮g/ml kanamycin and
induced at an A600 of 0.7–0.85 with 0.1 mM isopropyl-␤-D-thio-
galactopyranoside (IPTG).3 After 4 h, cells were harvested by
centrifugation for 25 min at 3000 ϫ g. Large scale cell cultiva-
tion was conducted in the Biotechnology Resource Center,
University of Minnesota. A 440-liter culture was prepared in a
550-liter DCI bioreactor (DCI-Biolafitte, St. Cloud, MN) using
a Rhapsody digital controller system and induced with 0.5 mM
IPTG. Cells were harvested, lyophilized, and then stored at
Ϫ80 °C.
Purification of OleA—Cells were resuspended in 20 mM
sodium phosphate buffer, 500 mM NaCl, pH 7.4, with EDTA-
free protease inhibitor tablets (Roche Applied Science). Cells
were disrupted by three passes through a chilled French pres-
sure cell at 1200 p.s.i. and centrifuged at 27,000 ϫ g for 90 min
to obtain the soluble protein fraction. The soluble fraction was
centrifuged at 27,000 ϫ g for 30 min to clear, prior to loading
onto an Amersham Biosciences LCC 501 FPLC equipped with a
5-ml Ni(II)-loaded HisTrap HP column (Amersham Biosci-
ences) equilibrated with 20 mM sodium phosphate, 500 mM
NaCl, pH 7.4, buffer. The OleA protein was eluted at 135 mM
imidazole. Ten g, wet weight, of cell paste yielded 60 mg of
purified OleA. Fractions were analyzed by SDS-PAGE and Sim-
ply Blue Safestain (Invitrogen). Pooled fractions were concen-
trated, and imidazole was removed, with three passes through a
50-ml pressure concentrator (Amicon) using a 10,000 molecu-
lar weight cut-off membrane (Millipore). Alternatively, after
concentration of fractions, the OleA protein was dialyzed three
EXPERIMENTAL PROCEDURES
Chemical Synthesis and Analysis—␤-Ketocarboxylic acid
syntheses have been previously reported (15), but the literature
does not describe the synthesis of higher benzyl esters of
␤-ketocarboxylic acids derived from fatty acids. The detailed
procedure used for the synthesis of 2-myristoylmyristic acid
(2-dodecyl-3-ketohexadecanoic acid) is described in the
supplemental material.
In brief, the forced Claisen condensation method described
by Briese and McElvain (16, 17) was adapted to the coupling of
benzyl myristate, using a half-equivalent of sodium benzyl alco-
holate in benzyl alcohol as the basic promoter, removing benzyl
alcohol by heating under vacuum to force the condensation
nearly to completion. Benzyl 2-myristoylmyristate (benzyl
2-dodecyl-3-ketohexadecanoate) so prepared (70% yield and
ϳ90% purity after bulb-to-bulb distillation) crystallized, mp
34.5–35.8 °C. The purity of benzyl 2-myristoylmyristate was
estimated from NMR data in CDCl3 solution, which show no
conclusive evidence for tautomeric enolic forms being present.
3 The abbreviations used are: IPTG, isopropyl-␤-D-thiogalactopyranoside;
DTNB, 5,5Ј-dithio-bis-(2-nitrobenzoic acid); FID, flame ionization detec-
tion; ESI, electrospray ionization.
2 Myristoyl is equivalent to tetradecanoyl.
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Purification and Characterization of OleA
times at 4 °C to remove the imidazole. Protein concentrated up for 5 min in either 1 ml or 250 ␮l. DTNB was incubated
to 30 mg/ml remained soluble and active.
with the reaction mixture for 2 min and quantified
Identification and Expression of Active OleD—Using the spectrophotometrically.
NCBI Blast algorithm (18), oleD genes were identified.
Hydrocarbon Detection Enzyme Assay—A glass vial contain-
Sequences from Chloroflexus auranticus (Caur_3530), ␥-pro- ing a 250-␮l total volume of 100 mM Tris, pH 7.4, with 200–600
teobacterium NOR5-3 (ZP_05127041.1), Xylella fastidiosa ␮g of OleA, 5–25 ␮g of OleD, 66 ␮g of OleC, 1.4 mM NADPH,
Temecula1 (NP_779252.1), and X. campestris spv. campestris 8 mM MgCl2, 3 mM ATP, and 1.2 mM myristoyl-CoA or an
str. ATCC 33913 (NP_635614.1) were optimized for expression excess of 14-heptacosanone was incubated overnight at 30 °C
in E. coli (see supplemental Fig. S2) and cloned into pJexpress with gentle shaking. Products were extracted with 250 ␮l of
expression vectors with a T7 promoter by DNA 2.0. Vectors ethyl acetate using 16-hentriacontanone ketone (Tokyo Kasei
were transformed into E. coli One Shot BL21 (DE3) (Invitro- Kogyo Co., Ltd.) as an internal standard. After vortexing and 5
gen). Proteins were screened for activity and expression using min of gentle centrifugation, the top solvent layer was trans-
50-ml LB cultures with 50 ␮g/ml kanamycin. Cells were ferred to a glass vial and analyzed using a gas chromatograph
induced with 0.1 mM IPTG at an A600 of 0.55–0.75. Soluble cell equipped with a flame ionization detector HP 7890A (Hewlett
extracts were combined with OleA, OleC, and cofactors to test Packard, Palo Alto, CA) and mass spectrometer HP 5975C
for the production of alkenes using the GC-MS enzyme assay (GC-MS-FID). GC was conducted under the following condi-
described below. The OleD protein originating from X. camp- tions: helium gas, 1.75 ml/min; HP-1ms column (100% dimeth-
estris was the only protein found to support alkene biosynthesis. ylsiloxane capillary; 30 m ϫ 250 ␮m ϫ 0.25 ␮m); temperature
Cultures were scaled up using 2-liter flasks containing 500 ml of ramp, 100–320 °C; 10 °C/min, hold at 320 °C for 5 min, 250 °C
LB with 50 ␮g/ml kanamycin. Cultures were grown at 37 °C injection port, and split at the outlet between MS and FID. The
with agitation at 225 rpm. The culture was induced at an A600 of mass spectrometer was run under the following conditions: elec-
0.7–0.8 with 0.1–0.4 mM IPTG and grown at 30 °C shaking at tron impact at 70 eV and 35 ␮A. The flame ionization detector was
225 rpm for 20 h. Cells were harvested by centrifugation for 25 set at 250 °C with hydrogen flow set at 30 ml/min, air set at 400
min at 3000 ϫ g.
ml/min, andheliummakeupgassetat25ml/min. GC-MSwasalso
Purification of OleC and OleD and Assay of OleD—The clon- used as described (10) for detecting ketones derived from sponta-
ing, expression, and purification of OleC was described previ- neous decarboxylation of the OleA ␤-keto acid products, using
ously (19). For purification of OleD, cell pellets were resus- 200–600 ␮g of OleA and 1 mM acyl-CoA substrates.
pended in 20 mM sodium phosphate, 500 mM NaCl, pH 7.4, with
Radiolabeled Acyl-CoA Assay—Reactions of 200 ␮l included
EDTA-free protease inhibitor tablets (Roche Applied Science) 0.2 ␮Ci of [1-14C]myristoyl-CoA (40–60 mCi/mmol; Ameri-
and passed through a chilled French pressure cell three times at can Radiolabeled Chemicals (St. Louis, MO)), 750 ␮M myris-
1200 p.s.i. The cell lysate was centrifuged at 27,000 ϫ g for 90 toyl-CoA, and 1 mg of OleA in 100 mM Tris, pH 7.4. Samples
min, and the soluble fraction was centrifuged for an additional were analyzed using high pressure liquid chromatography
30 min. The soluble fraction was passed though a 0.20-␮m (HPLC) on a Shimadzu HPLC system equipped with a UV
syringe filter prior to chromatography. A 5-ml Ni(II)-loaded detector (Shimadzu, Columbia, MD) and a ␤-ram radioflow
HisTrap HP column (Amersham Biosciences) equilibrated detector operated with the Laura 4 data acquisition/evaluation
with 20 mM sodium phosphate, 500 mM NaCl, pH 7.4, buffer software (IN/US Systems, Tampa, FL). UV detection was set at
was used for purification. Alternatively, 50 mM MOPS, 1% 259 or 274 nm. Unfiltered samples of 50- or 100-␮l volume were
Tween 20, pH 7.0, was used for purification to improve solubil- injected onto an analytical reverse phase Alltima HPC8 column
ity. Fractions were analyzed for purity by SDS-PAGE and Sim- with 5-␮m packing (Alltech 250 ϫ 4.6 mm) and a C8 guard
ply Blue Safestain (Invitrogen). Fractions eluting at 450 and 500 column. The column was equilibrated in 50% 20 mM ammo-
mM imidazole were pooled. The pooled protein was concen- nium acetate, pH 5.4 (A) and 50% 85:15 acetonitrile/methanol
trated using a 50-ml pressure concentrator (Amicon) with a (B) and the following method adapted from Ref. 22. Linear gra-
10,000 molecular weight cut-off membrane (Millipore) and dia- dients were as follows: 50% A, 50% B, 0–10 min; ramp to 70% B,
lyzed three times at 4 °C. After dialysis, protein was centrifuged 10–15 min; 70% B, 15–30 min; ramp to 100% B, 30–35 min;
at 14,000 ϫ g for 15 min at 4 °C to remove precipitated protein. 100% B, 35–50 min; return to 50% A, 50% B, 50–55 min; and
OleD was previously suggested to be a ketone reductase (13). equilibrate, 55–70 min. The flow rate was 1 ml/min. The scin-
It was shown here to be active in a 250-␮l reaction mixture tillant (Monoflow X; National Diagnostics, Atlanta, GA) flow
consisting of 100 mM Tris, pH 7.4, containing OleA, OleD, rate was 3 ml/min.
OleC, 8 mM MgCl2, 80 ␮M ATP, 260 ␮M myristoyl-CoA, and
120 ␮M NADPH. NADPH oxidation was followed spectropho- matically produced and synthetic ␤-keto acid was performed
tometrically at 340 nm. using an LCQ-classic (Thermo Fisher Scientific) ion trap mass
Mass Spectrometry Analysis—Mass spectrometry on enzy-
Detecting the Release of CoASH Thiol for Assay of OleA Sub- spectrometer with electrospray ionization (ESI) mode. Samples
strate Range—Release of the free thiol group of CoASH was were introduced by loop injection of 5 ␮l. Product ion spectra
detected by the addition of 5,5Ј-dithio-bis-(2-nitrobenzoic for 2-myristoylmyristic acid, m/z 437 (M Ϫ H), was identified in
acid) (DTNB) measured spectrophotometrically at 412 nm negative ion mode, as well two additional ions, m/z 393 and m/z
(⑀412 ϭ 13,600 MϪ1 cmϪ1) (20, 21). Acyl-CoA substrates, pur- 473/475. The fatty acid HPLC peak was analyzed by direct infu-
chased from Sigma-Aldrich, were reacted with OleA protein sion into a Quantum Discovery Max (Thermo Finnigan) mass
Ϫ
in 100 mM Tris, pH 7.4, and incubated at room temperature spectrometer operated in negative ion mode. ESI -MS spectra
10932 JOURNAL OF BIOLOGICAL CHEMISTRY
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Purification and Characterization of OleA
General Characteristics of OleA—The subunit molecular
weight of the native OleA protein is 36,629, but, as engineered
here with the His tag, it is 38,792. The protein migrates some-
what higher than this on SDS-PAGE (Fig. 2B). The OleA pro-
tein subunit Mr is near the middle of the range found in homol-
ogous proteins from the thiolase superfamily (Table 1). The
Mycobacterium Pks13 protein is a large multidomain protein
with the condensing enzyme domain defined as 44,122 in the
annotation on the NCBI server. The native molecular weight of
OleA was estimated to be 62,000 by gel filtration chromatogra-
phy. This is suggestive of a subunit stoichiometry of 2 for the
native enzyme. Hydroxymethylglutaryl-CoA reductase, FabH,
and the Mycobacterium Pks13 are all dimers, and the Zoogloea
thiolase is a tetramer (23–26).
The OleA protein shows a low sequence relatedness with
homologous proteins in the thiolase superfamily (Table 1).
With such divergence, it is not surprising that the cellular func-
tions of the proteins are quite different. However, the general
biochemical reaction catalyzed by all of the enzymes shown in
Table 1 involves the condensation of acyl substrates. These
condensation reactions occur by either a decarboxylative or
non-decarboxylative (27) mechanism, and the results described
below are consistent with a non-decarboxylative mechanism
for OleA. In both cases, thiolase superfamily proteins use a con-
served active site cysteine and generate an acyl enzyme inter-
mediate (28). OleA shares this conserved active site cysteine
residue (Table 1). In the vicinity of the conserved cysteine, the
amino acids in the OleA from X. campestris and Micrococcus
luteus (11) are highly conserved (Table 1).
FIGURE 2. SDS-polyacrylamide gel showing standard molecular weight
markers (A) and purified OleA (B).
for myristic acid was m/z 227 (M Ϫ H). Electron impact mass
spectrometry in conjunction with GC (GC-MS) was performed
to obtain the spectra of the ␤-keto acid derivatized with diazo-
methane. The solvent was evaporated with N2 and then taken
up in methyl-t-butyl-ether to run on GC-MS as described pre-
viously (10). The ketone molecular ion, m/z 394, was also iden-
tified by this method.
Initial Defining of Substrate Specificity and Reaction Products
of OleA—Enzymes catalyzing condensation or hydrolysis reac-
tions with acyl-CoA substrates release coenzyme A that can be
assayed colorimetrically using DTNB (20, 29–31). Both pro-
posed mechanisms (Fig. 1, A and B) showed OleA-catalyzed
coenzyme A release, and this assay was used to monitor enzyme
activity during purification. With purified OleA, DTNB was
used to determine the stoichiometry of coenzyme A formation
and to begin to discern the substrate specificity of OleA.
First, the coenzyme A product stoichiometry was deter-
mined using either myristoyl-CoA or palmitoyl-CoA and
allowing the substrate to completely react. With either sub-
strate, the reaction stoichiometry was 1.0 mol of coenzyme A
released/mol of acyl-CoA consumed. In this manner, acyl-CoA
chains of different lengths were tested with OleA using a time of
Analytical Gel Filtration—A Superdex 75 10/100 GL (Amer-
sham Biosciences) size exclusion column was used on an AKTA
(General Electric) FPLC with elution at 0.5 ml/min. The col-
umn was equilibrated with 20 mM sodium phosphate, 500 mM
NaCl, pH 7.4. Molecular weight standards (Bio-Rad) were used
with a range of 1350–670,000 to create a standard curve. Three
additional standards were used in a closer Mr range to where
OleA eluted, chymotrypsin (Mr ϭ 25,000), albumin (Mr ϭ
67,000), and conalbumin (Mr ϭ 77,000).
RESULTS
Cloning and Expression of oleA Genes and Purification of incubation in which palmitoyl-CoA reacts completely as
OleA Protein—The oleA genes from C. litoralis KT71, X. camp- described under “Experimental Procedures.” Under those con-
estris spv. campestris str. ATCC 33913, X. fastidiosa 9a5c, ditions (Table 2), palmitoyl-CoA reacted more completely than
P. pacifica SIR-1, and ␥-proteobacterium NOR5–3 were each myristoyl-CoA. Octanoyl-, decanoyl-, lauroyl-, palmitoleoyl-,
cloned into E. coli and tested for the production of soluble OleA and stearoyl-CoA were also found to undergo reaction to
protein. The recombinant E. coli containing the X. campestris release coenzyme A (Table 2), but acetyl-CoA did not.
gene showed a high amount of soluble OleA protein, as deter-
Subsequent experiments were conducted to examine if coen-
mined by SDS-PAGE, and was found to be active in purified zyme A was formed as a consequence of acyl-group condensa-
form, and that strain was therefore selected for further studies. tion, thioester bond hydrolysis, or some mixture of the two
E. coli cells expressing a His-tagged X. campestris OleA protein reactions. Based on previous observations (11, 13, 14), it was
were grown in a 550-liter bioreactor vessel, harvested, and known that long-chain ketones were the observed condensa-
lysed. Following chromatography on a nickel column, the pro- tion products. In subsequent experiments in this study, it was
tein was shown to be homogenous, as indicated by SDS-PAGE shown that ␤-keto acids are the initial products, and those
(Fig. 2B).
decarboxylate quantitatively to the corresponding ketone. In
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Purification and Characterization of OleA
TABLE 1
Properties of OleA compared with homologous proteins in the thiolase superfamily
Sequence Calculated Calculated
Protein (organism)a
Accession number identityb
Mr
pI
Cellular function
Claisen mechanism
Sequence signaturec
%
OleA (X. campestris)
OleA (M. luteus) (11)
NP_635607
100
36,629
5.6
Alkene biosynthesis
Proposed
Non-decarboxylative
NACLAFING
YP_002957382.1
38
19
16
36,653
40,416
43,204
4.8
5.9
5.0
Alkene biosynthesis
PHB biosynthesis
Proposed decarboxylative NACLGFVNG
Thiolase (Zoogloea ramigera) (23) AAA27706.1
Non-decarboxylative
Non-decarboxylative
QLCGSGLRA
EACYAATPA
HMG-CoA synthase (Homo
sapiens) (26)
1XPL_A
Mevalonate pathway
FabH (E. coli) (25)
1EBL_A
24
19
33,523
44,122
5.1
5.2
Fatty acid biosynthesis
Decarboxylative
AACAGFTYA
TACSSSLVA
Mycobacterium Pks13d (24)
CAA17864
Mycolic acid biosynthesis Decarboxylative
a Numbers in this column represent references from which the data in the table were obtained.
b Via Needleman-Wunch and BLAST algorithms and comparison with OleA from X. campestris.
c Amino acid sequence surrounding the active site cysteine conserved in thiolase superfamily proteins.
d Alignment to keto-acyl synthase domain only, as defined by NCBI.
TABLE 2
TABLE 3
Substrate specificity of OleA as determined by CoA release
Values shown are the average of triplicate determinations with S.E.
Product ratios determined by GC-MS for reactions of OleA with acyl-
CoA substrates of different carbon chain lengths as indicated by the
left-hand column and the top row
The products, ketones (Cx) and fatty acids (FAx), are indicated in order of decreasing
abundance as determined by peak area integration as described under “Experimen-
tal Procedures.” The observed partitioning between condensation of similar or dif-
ferent chains or acyl-CoA hydrolysis is illustrated at the bottom.
Substrate
Carbon chain
Percentage of
CoA producta theoretical yieldb
Common name
length
␮M
%
Palmitoyl-CoA
Myristoyl-CoA
Lauroyl-CoA
16
14
12
65.0 Ϯ 0.9
63.2 Ϯ 0.4
51.4 Ϯ 1.9
36.9 Ϯ 0.9
27.2 Ϯ 1.6
18.7 Ϯ 1.8
8.0 Ϯ 2.2
NDc
100
97
79
Palmitoleoyl-CoA 16
57
Decanoyl-CoA
Stearoyl-CoA
Octanoyl-CoA
Acetyl-CoA
10
18
8
42
29
12
2
ND
a Free coenzyme A detected as described under “Experimental Procedures.”
b Starting substrate was 65 ␮M; 65 ␮M product is 100% of theoretical yield.
c ND, no detectable activity.
this context, reaction mixtures were solvent-extracted and sub-
jected to GC-MS to identify ketones derived from condensation
and/or fatty acids derived from acyl chain hydrolysis.
Previous in vivo experiments identified asymmetric ketones,
indicating that fatty acyl chains of different chain lengths could
be condensed (13). In this context, experiments were con-
ducted with mixtures of fatty acyl-CoA substrates. All pairwise
combinations of saturated C10, C12, C14, and C16 and C16 mono-
unsaturated (C16:1) acyl-CoA substrates were incubated,
extracted, and analyzed for products by GC-MS and GC-FID.
In all, 15 product mixtures were analyzed. The results are shown
in Table 3. It was found that acyl-CoA hydrolysis to the corre-
sponding fatty acid was a major reaction in most cases. Only with
C
12 acyl condensation and C14 plus C16:1 condensation were the eluting at 44.3 min (compound 2) was analyzed by GC-MS and
majorproductsderivedfromacondensationoffattyacylchains. In found to be 14-heptacosanone, but more of the radioactivity
the case of C14 condensations (myristoyl-CoA), the ketone co-migrated with a more polar product eluting at 40.0 min (Fig.
14-heptacosanone was produced at only slightly lower levels than 3). The major peak eluting at 40.0 min (compound 1) showed
the hydrolysis product, myristic acid.
very little absorbance at 259 nm, consistent with the absence of
The identification of ketones as the condensation products a coenzyme A moiety. Over time, the peak at 40 min diminished
by use of GC-MS led to the question of whether the decarbox- with a concomitant increase in the peak at 44.3 min (Fig. 3,
ylation was enzymatic or whether the decarboxylation occurs inset). This observation was consistent with a decarboxylation
due to the labile nature of the reaction intermediate preceding of the compound at 40.0 min giving rise to increasing concen-
the formation of the ketone. Further investigations were con- trations of 14-heptacosanone over the course of 6.5 h. This was
ducted using a C1-labeled acyl-CoA substrate to track the car- also indicated because the compound eluting at 44.3 min con-
boxyl carbon.
tained only half of the 14C as did the compound at 40 min,
Identification of Initial Condensation Product—OleA reac- consistent with a loss of a carbon atom as carbon dioxide.
tions with [1-14C]myristoyl-CoA were analyzed using HPLC Moreover, ␤-keto acids are known to be labile, and the decar-
fitted with a radioflow detector. A major peak eluting at 22.4 boxylation of 2-myristoylmyristic acid would be expected to
min was identified as myristic acid (Fig. 3). The HPLC peak produce 14-heptacosanone.
10934 JOURNAL OF BIOLOGICAL CHEMISTRY
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Purification and Characterization of OleA
FIGURE 3. OleA reaction products with [14C]myristoyl-CoA as the sub-
strate. A, HPLC profile showing radioactive peaks. Inset, plot of the radioac-
tivity detected in product 1 and product 2 over the course of 6 h when a
reaction mixture was incubated at room temperature. B, schematic of the
reactions leading to the formation of product 1 and product 2.
To investigate this further, the benzyl ester of 2-myris-
toylmyristic acid was synthesized. It was hydrogenolyzed with
palladium and hydrogen to produce 2-myristoylmyristic acid.
This latter compound was observed to undergo rapid decarbox-
ylation to produce 14-heptacosanone.
FIGURE 4. Mass spectra for products from the reaction of diazomethane
with product 1 (40 min retention time) from the reaction of OleA with
2-myristoylmyristic acid (A) and synthetic 2-myristylmyristic acid (B).
TABLE 4
Treatment of synthetic 2-myristoylmyristic acid with diazo-
methane yielded the methyl ester. The synthetic methyl ester
was compared with the enzyme-produced compound collected
at 40.0 min that had been immediately reacted with diazometh-
ane. Both methylated compounds showed a GC retention time
of 20.6 min and essentially identical mass spectra (Fig. 4). The
parent ion at m/z 452 is present in both, but it is a minor ion. In
this context, electrospray ionization mass spectrometry was
conducted on the free acid product 1 from the OleA reaction
with myristoyl-CoA (Table 4, top) and the synthetic standard
2-myristoylmyristic acid (Table 4, bottom). In this case, a major
negative ion was observed (m/z 437) with a mass of 1 Da less
than the molecular mass of 2-myristoylmyristic acid in both the
biological product and the standard. A second major fragment
of m/z 393 found in both is consistent with the loss of carbon
dioxide in the mass spectrometer. Another ion fragment was
detected at m/z 473/475, suggested to be [M Ϫ H ϩ HCl].
Role of OleA in Olefin Biosynthesis—OleA has been proposed
to function with other Ole proteins to produce olefins (11, 13,
14). Other Ole proteins were purified as described under
“Experimental Procedures” and tested in admixture with OleA
and with myristoyl-CoA as the substrate. Gas chromatography-
mass spectrometry was used because it can detect both the
OleA product following its decarboxylation to 14-heptaco-
sanone and the expected olefin 14-heptacosene if the entire
ESI mass spectrometry of product 1 from reaction of OleA with myris-
toyl-CoA as shown in Fig. 3 and synthetic 2-myristoylmyristic acid
Molecular
ESI, negative ion mode m/z
Sample
mass
(intensity)
Da
Product 1
2-Myristoylmyristic acid
393 (52), 437 (29), 473 (19)
393 (45), 437 (36), 473 (19)
438
FIGURE 5. Gas chromatogram with MS detector showing products
observed upon co-incubations with OleA and OleC (foreground trace)
and OleA, OleC, and OleD (background trace).
biosynthetic pathway were functional. Fig. 5 shows that OleA observed in addition to the peak corresponding to 14-heptaco-
and OleC in admixture produced only 14-heptacosanone (elu- sanone. The identities of the products were confirmed by mass
tion time 21.8 min), the product observed with OleA alone. spectrometry.
However, when OleA was incubated with myristoyl-CoA,
To directly demonstrate that 2-myristoylmyristic acid was
OleC, and OleD, the olefin 14-heptacosene (20.4 min) was the intermediate giving rise to the olefin, we incubated syn-
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Purification and Characterization of OleA
Next, it was tested if the ketone 14-heptacosanone could also
give rise to 14-heptacosene or any other discernible product. In
this experiment (Fig. 6B), OleC and OleD were incubated with
14-heptacosanone under the same conditions as described
above, and the olefinic product 14-heptacosene was not
detected. A minor peak was observed at 20.35 min that had a
mass spectrum different than that of 14-heptacosene (m/z 356).
The small 20.35 min peak was also present in incomplete reac-
tion mixtures that lacked OleD (Fig. 6C), further demonstrating
that it is a contaminant and not relevant to olefin biosynthesis.
It was found to be a minor impurity in the synthetic ketone
preparation.
Overall, these data suggest that X. campestris OleA produced
␤-ketoacid intermediates from acyl-CoAs (C8–C18) and that
the ketone is a non-physiological product arising from sponta-
neous decarboxylation. Note that ketones have been observed
in vivo in recombinant bacteria containing heterologous oleA
genes (11, 13). Additionally, Albro et al. (8) incubated ketones
with crude protein fractions and failed to observe olefins. How-
ever, when a full suite of ole genes are present in native hosts,
olefinic products, and not ketones, are typically observed (12).
Thus, previous in vivo results are fully consistent with the in
vitro data obtained in the present study.
DISCUSSION
In this study, the OleA protein from X. campestris was puri-
fied to homogeneity and shown to condense fatty acyl-CoA
substrates to produce a condensed ␤-ketoacid with the release
of 2 mol of CoA. The ␤-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 (11) 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 (18), 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 ␤-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 (11). In the present study,
the recombinant E. coli strain expressing the X. campestris
OleA protein alone was also observed to produce long-chain
FIGURE 6. Gas chromatogram and accompanying mass spectra of peaks
eluting between 20.3 and 20.4 min from extract of reaction mixture con-
taining OleC and OleD incubated with 2-myristoylmyristic acid (A),
extract of reaction mixture containing OleC and OleD incubated with
14-heptacosanone (B), and extract of reaction mixture containing OleC
incubated with 14-heptacosanone (C).
thetic 2-myristoylmyristic acid with OleC and OleD. The ketones that were not observed in the wild-type E. coli (data not
experiment yielded 14-heptacosanone and the expected ole- shown). The in vitro data in this study showed that the ketones
finic product from head-to-head condensation, 14-hepta- readily arise from the decarboxylation of a corresponding ␤-ke-
cosene (Fig. 6, peak A, 20.4 min). The identity of the compound toacid intermediate. These observations are all consistent with
was confirmed by the mass spectrum shown above the GC a non-decarboxylative Claisen condensation as shown in Fig.
chromatogram in Fig. 6A, with the major ion, m/z 378, repre- 1B and difficult to reconcile with the proposed decarboxylative
senting the molecular ion.
reaction shown in Fig. 1A.
10936 JOURNAL OF BIOLOGICAL CHEMISTRY
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Purification and Characterization of OleA
hydrolysis to yield the final ␤-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 stoi-
chiometry in which 2 mol of coenzyme A are observed/mol of
condensed product. Second, the observed high rate of hydroly-
sis of acyl-CoAs to produce fatty acids is not unexpected if the
enzyme has a mechanism to hydrolyze thioester-linked inter-
mediates 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 (27, 28),
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 7. Proposed 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 ␤-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 dem-
onstrated here. Second, the suggested generation of a carban-
ion (Fig. 7B) requires a general base that remains to be identi-
fied. 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.
The reaction catalyzed by OleA is somewhat reminiscent of
the Zoogloea thiolase reaction that catalyzes the first step in the
biosynthesis of polyhydroxybutyrate (23). In the latter reaction,
however, the condensed product is a ␤-ketoacetyl-CoA, aceto-
acetyl-CoA, and with OleA, the product is a ␤-keto acid. Several
lines of evidence strongly suggested that OleA does not pro-
duce a ␤-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 hydrocar-
bons. 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-myris-
toylmyristoyl-CoA, and this was never detected.
This study identified the product of the OleA-catalyzed reac-
tion to be a ␤-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 ␤-ketoacid intermediate generated by OleA. This was sup-
ported 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 intermedi-
ate. 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.
Based on the data obtained and the known role of the con-
served cysteine found in other members of the thiolase super-
family, 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 teth-
ered substrate is probably activated by an active site base to
yield a carbanion on the tethered substrate (Fig. 7C). The car-
banion 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
Acknowledgments—We thank David Sukovich for providing a selec-
tion of oleA genes used in these studies. We acknowledge Fred Schen-
del and Mary Pruss for fermentation and cell harvesting. We espe-
cially 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 Spec-
trometry and Proteomics and Mass Spectrometry Services in the
Masonic Cancer Center with the assistance of Tom Krick, Brock Mat-
ter, and Peter Villalta.
APRIL 1, 2011•VOLUME 286•NUMBER 13
JOURNAL OF BIOLOGICAL CHEMISTRY 10937

Purification and Characterization of OleA
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10938 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 13•APRIL 1, 2011

Enzymology:
Purification and Characterization of OleA
from Xanthomonas campestris and
Demonstration of a Non-decarboxylative
Claisen Condensation Reaction
Janice A. Frias, Jack E. Richman, Jasmine S.
Erickson and Lawrence P. Wackett
J. Biol. Chem. 2011, 286:10930-10938.
doi: 10.1074/jbc.M110.216127 originally published online January 25, 2011
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