Functional characterization of an NADPH dependent 2-alkyl-3-ketoalkanoic acid reductase involved in olefin biosynthesis in stenotrophomonas maltophilia

Article abstract of DOI:10.1021/bi201096w

OleD is shown to play a key reductive role in the generation of alkenes (olefins) from acyl thioesters in Stenotrophomonas maltophilia. The gene coding for OleD clusters with three other genes, oleABC, and all appear to be transcribed in the same direction as an operon in various olefin producing bacteria. In this study, a series of substrates varying in chain length and stereochemistry were synthesized and used to elucidate the functional role and substrate specificity of OleD. We demonstrated that OleD, which is an NADP(H) dependent reductase, is a homodimer which catalyzes the reversible stereospecific reduction of 2-alkyl-3-ketoalkanoic acids. Maximal catalytic efficiency was observed with syn-2-decyl-3-hydroxytetradecanoic acid, with a k_cat/K_m 5- and 8-fold higher than for syn-2-octyl-3- hydroxydodecanoic acid and syn-2-hexyl-3-hydroxydecanoic acid, respectively. OleD activity was not observed with syn-2-butyl-3-hydroxyoctanoic acid and compounds lacking a 2-alkyl group such as 3-ketodecanoic and 3-hydroxydecanoic acids, suggesting the necessity of the 2-alkyl chain for enzyme recognition and catalysis. Using diastereomeric pairs of substrates and 4 enantiopure isomers of 2-hexyl-3-hydroxydecanoic acid of known stereochemistry, OleD was shown to have a marked stereochemical preference for the (2R,3S)-isomer. Finally, experiments involving OleA and OleD demonstrate the first 3 steps and stereochemical course in olefin formation from acyl thioesters; condensation to form a 2-alkyl-3-ketoacyl thioester, subsequent thioester hydrolysis, and ketone reduction.

Full text of DOI:10.1021/bi201096w

Article
pubs.acs.org/biochemistry
Functional Characterization of an NADPH Dependent
-Alkyl-3-ketoalkanoic Acid Reductase Involved in Olefin
Biosynthesis in Stenotrophomonas maltophilia
2
Shilah A. Bonnett,† Kancharla Papireddy, Samuel Higgins, Stephen del Cardayre,
,‡
†,‡
†
§
,†
and Kevin A. Reynolds*
†Department of Chemistry, Portland State University, Portland, Oregon 97201, United States
§LS9, Inc., San Francisco, California 94080, United States
*
S Supporting Information
ABSTRACT: OleD is shown to play a key reductive role in
the generation of alkenes (olefins) from acyl thioesters in
Stenotrophomonas maltophilia. The gene coding for OleD
clusters with three other genes, oleABC, and all appear to be
transcribed in the same direction as an operon in various olefin
producing bacteria. In this study, a series of substrates varying
in chain length and stereochemistry were synthesized and used
to elucidate the functional role and substrate specificity of
OleD. We demonstrated that OleD, which is an NADP(H)
dependent reductase, is a homodimer which catalyzes the reversible stereospecific reduction of 2-alkyl-3-ketoalkanoic acids.
Maximal catalytic efficiency was observed with syn-2-decyl-3-hydroxytetradecanoic acid, with a k /K 5- and 8-fold higher than
cat
m
for syn-2-octyl-3-hydroxydodecanoic acid and syn-2-hexyl-3-hydroxydecanoic acid, respectively. OleD activity was not observed
with syn-2-butyl-3-hydroxyoctanoic acid and compounds lacking a 2-alkyl group such as 3-ketodecanoic and 3-hydroxydecanoic
acids, suggesting the necessity of the 2-alkyl chain for enzyme recognition and catalysis. Using diastereomeric pairs of substrates
and 4 enantiopure isomers of 2-hexyl-3-hydroxydecanoic acid of known stereochemistry, OleD was shown to have a marked
stereochemical preference for the (2R,3S)-isomer. Finally, experiments involving OleA and OleD demonstrate the first 3 steps
and stereochemical course in olefin formation from acyl thioesters; condensation to form a 2-alkyl-3-ketoacyl thioester,
subsequent thioester hydrolysis, and ketone reduction.
iochemical pathways in which alkanes or alkenes are
generated from the CoA activated fatty acids are known
genomes contain all four ole synthesis genes, 7 of which
10,16
B
contained a fused oleBC gene pair.
In all cases, the four ole
and are of significant interest for generation of diesel and
gasoline type products. Alkanes can be generated from an acyl-
CoA thioester, a well characterized process which involves reduc-
tion to the corresponding aldehyde and a subsequent decarbon-
genes are clustered together and appear to be transcribed in the
same direction as an operon.
Initial in vitro and in vivo analysis demonstrated that OleA
catalyzes a decarboxylative condensation of two activated
1
7,9,10,16
ylation step. There have also been reports dating back over
fatty acids to give a ketone.
More recently, OleA from
4
0 years of alkenes (olefins) with varying chain length (C −C )
X. campestris was shown to catalyze the nondecarboxylative
condensation of two CoA activated fatty acids, releasing
two molecules of CoA and providing a 2-alkyl-3-ketoalkanoic
23
33
being produced by various organisms such as Stenotrophomonas
maltophilia, Kineococcus radiotolerans, Shewanella oneidensis,
Xanthomonas campestris, and various Micrococcus, Arthrobacter,
and Chloroflexus species. The formation of olefins is thought
to occur through a process involving the head-to-head
condensation of two activated fatty acids followed by β-keto
group reduction. The resulting olefin contains a double bond
near the middle of the chain, presumably located at the sight
9
acid as the likely final product (Figure 1). This acid then
2−9
decarboxylates either in vitro or under GC−MS analysis
9
conditions to the corresponding ketone. In vitro and in vivo
experiments involving OleC and OleD demonstrated their
involvement in the subsequent steps following the OleA
catalyzed condensation. While these studies have provided the
first clear biochemical insight into the role of OleA in the
processes of olefin generation, the specific role of the remaining
proteins OleB−D, remains unknown.
2−6,10−15
of condensation.
holds a potential for generating renewable petroleum from
microbial fermentation, but is poorly understood.
This reductive condensation process
A series of genetic and biochemical investigations of the olefin
pathway resulted in the identification of four genes, annotated
oleABCD, to be responsible for long chain alkene biosynthesis in
Received: July 15, 2011
Revised: September 16, 2011
Published: September 29, 2011
7−10,16
olefin producing organisms.
Approximately, 70 bacterial
©
2011 American Chemical Society
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Figure 1. Working hypothesis for the roles of OleA−D in catalyzing olefin formation from two thioester activated fatty acids. R and R are variable;
1
2
X = CoA or acyl carrier protein.
Our efforts have been focused on the functional and
biochemical characterization of OleD, which has been annotated
as an NAD(P)H dependent enzyme and belongs to the short
chain dehydrogenase/reductase superfamily (SDR). Members of
were >90% pure, based on SDS−PAGE analysis and were
stored in 50 mM Tris·HCl (pH 8.0), 100 mM NaCl, 10%
glycerol, and 1 mM TCEP at −80 °C.
Spectrophotometric Assay for OleD Activity. Enzy-
matic activity of OleD was monitored by change in absorbance
at 340 nm, due to the formation or consumption of NAD(P)H
+
this superfamily catalyze various NAD(P) /NAD(P)H depend-
ent reactions including oxidation, reduction, epimerization,
17−21
−1
−1
dehydrogenation, dehydration, and synthase reactions.
On
(ε340 = 6220 M cm ). Assays were performed in the
presence of 100 mM sodium phosphate (pH 7.5), 0.5% Tween
20, 1 mM TCEP, 0.5−1 mM substrate (suspended in DMSO
to give a final concentration of 1%), and either 0.2 mM
the basis of bioinformatic and preliminary biochemical
9,10
analysis,
OleD is believed to function as a reductase and
therefore potentially responsible for the reduction of 2-alkyl-3-
ketoalkanoic acids (Figure 1). To test this hypothesis, a series of
compounds were generated and used to probe the functionality
and substrate specificity, including stereochemistry and carbon
chain length, of OleD. The results of these analyses and the
roles of OleA and OleD in the first two steps of the olefin
biosynthetic pathway are presented.
+
NAD(P)H or 1 mM NAD(P) in a final volume of 0.5 mL. The
reactions were initiated upon the addition of protein (0.05−
1 μM) and allowed to proceed for 10 min at 25 °C. All assays
were performed in triplicate.
Fluorescence Assay for OleD Activity. A modified
method for the fluorescence detection of alkaline NAD(P)+
EXPERIMENTAL PROCEDURES
Table 1. Summary of the Compounds Utilized in the
Biochemical Analysis of S. maltophilia OleD and Their
Relative Activity
■
Materials. All chemicals were from Sigma or Fisher unless
+
+
otherwise noted. β-NADH, β-NAD , β-NADPH, β-NADP ,
and dithiothreitol (DTT) were from Research Products
International Corp.; (3R/S)-3-hydroxydecanoic acid (45) was
from Matreya. ThioGlo-1, [10-(2,5-dihydro-2,5-dioxo-1H-
pyrrol-1-yl)-9-methoxy-3-oxo-methyl ester], was purchased
either from Calbiochem or Covalent Associates.
a
b
relative c
Substrate Synthesis and Characterization. The syn-
thetic strategies employed in the generation of various racemic
stereoisomers of 2-alkyl-3-ketoalkanoic acids and 2-alkyl-3-
hydroxyalkanoic acids, N-acetylcysteamine (SNAC) thioesters,
and methyl esters are outlined in the Supporting Information.
Using Evans aldol methodology, we have also synthesized all
four stereoisomers of 2-hexyl-3-hydroxydecanoic acid (17a,
compd
R1
R2
R3
stereochemistry activity (%)
9
n-C H
O
n-C H
15
(2R/S)
syn
13
0
6
13
7
1
1
1
1
2
6
7
8
9
0
n-C H9
−OH n-C H
4
5
11
15
19
anti
syn
0
n-C H
−OH n-C H
15
<1
20
<1
100
<1
ND
ND
0
6
13
7
anti
syn
n-C H
−OH n-C H
9
22,23
8
17
1
7b, 17c, and 17d).
All compounds were characterized by
anti
syn
NMR and liquid chromatography−mass spectrometry (LC−MS)
analysis and a summary of their structures and activity are
summarized in Table 1.
Expression and Purification of Recombinant K. radio-
tolerans OleA and S. maltophilia OleD His -Tag Fusion
Proteins. Expression plasmids carrying the oleD gene from
S. maltophilia and the oleA gene from K. radiotolerans cloned
into pET-21b vector system (Novagen) were a kind gift from
the renewable petroleum company LS9, Inc. (San Francisco,
n-C H
−OH n-C H
11 23
10
21
anti
syn
d
d
n-C H
−OH n-C H
13 27
12
25
anti
-
6
2
2
4
6
7
5
H
H
H
O
O
n-C H
5
11
15
15
n-C H
-
0
7
−OH n-C H
(3R/S)
0
7
a
For compounds 9 and 16−20 where the carboxylic acid was either a
NAC thioester or a methyl ester, no activity was observed.
CA). Each of the recombinant His -tagged fusion proteins were
b
6
Stereochemical assignment of syn ((2R,3S)- and (2S,3R)-enantio-
overproduced in Escherichia coli BL21(DE3) cells and purified
meric pair) and anti ((2S,3S)- and (2R,3R)-enantiomeric pair) was
based on NMR analysis and LC−MS comparison to enantiomerically
2
+
by Ni -agarose affinity chromatography using standard
protocols. Protein concentrations were determined using the
Qubit Fluorometer from Invitrogen. The resulting proteins
c
pure standards. Percent relative activity based on the comparison of
d
k
cat/K
values. ND denotes not determined due to solubility issues.
m
9
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2
4−26
in a 96 well plate was also used to examine OleD activity.
TCEP, 0.5% Tween 20, 1% DMSO, and either 0.2 mM
octanoyl-CoA, octanoyl-SNAC (44), (2R/S)-2-hexyl-3-ketode-
canoyl-SNAC (10), and/or sodium decanoate. Additional
analyses included addition of 0.5 mM NADPH and 2 μM
OleD. Samples were incubated for 90 min and analyzed by
LC−MS in the negative mode on a ThermoElectron LTQ-
Orbitrap high resolution mass spectrometer with a dedicated
Accela HPLC system using a Discovery HS C18 (250 × 2.1,
5 μm, Supelco) column at a flow rate of 0.3 mL/min. Buffer A
was 10 mM ammonium acetate in water and Buffer B was
10 mM ammonium acetate in acetonitrile. The following
solvent gradient was used in the analysis (time, % Buffer B):
0 min, 10%; 30 min, 100%; 35 min, 100%; 38 min, 10%; and
45 min, 10%.
Reactions were conducted under conditions described above
except concentrations of NAD(P)H up to 1 mM were used for
reduction reactions. At specific time points, 25 μL samples were
quenched with an equal volume of 0.3 M HCl and incubated
for 15 min at room temperature. Following the addition of
5 μL of 10 M NaOH, the samples were incubated for an
additional 2 h at room temperature in the dark. After the
addition of 180 μL of water, fluorescence was measured with an
excitation wavelength of 360 nm and an emission wavelength of
55 nm. A standard curve of NAD(P) was used to determine
product concentration. Sequential hydrolytic reductive activity
was also examined by fluorescence when 2 μM OleD, 1 μM
OleA, 0.25 mM (2R/S)-2-hexyl-3-ketodecanoyl-SNAC (10),
and 1 mM NADPH were incubated together.
Effects of DMSO and Nonionic Detergents on OleD
Activity. The effects of DMSO on enzyme activity was
examined by UV-spectroscopy in the presence of 100 mM
4
+
4
Fluorescence Based Assay for OleA Hydrolytic
Activity. A time course analysis of OleA hydrolytic activity
resulting in the formation of a free thiol (CoA or N-acetylcy-
steamine (SNAC) thioester) was carried out at 25 °C in the
sodium phosphate (pH 7.5), 1 mM TCEP, 0.5 mM syn-2-hexyl-
presence of 100 mM Tris-HCl (pH 8.0) and 10 mM MgCl ,
2
+
3
-hydroxydecanoic acid (17), 1 μM OleD, 1 mM NADP , and
0.1 mM TCEP, 1% DMSO, 0.5 μM OleA, and 0.5 mM of either
octanoyl-CoA, octanoyl-SNAC (44), or (2R/S)-2-hexyl-3-
ketodecanoyl-SNAC (10). A series of controls were conducted
in parallel, including the use of boiled protein, to ensure all
hydrolysis of acyl thioesters were due to enzymatic activity.
Aliqouts (50 μL) were withdrawn at specific time points and
added to a well of a black 96-well plate containing an equal
volume of DMSO to quench the reaction. After the addition
of 100 μL of 200 μM ThioGlo-1 solution (in DMSO) to each
well, the plate was incubated in the dark at room temperature
with gentle shaking for 20 min. All samples were analyzed by
fluorescence (excitation 378 nm and emissions at 480 nm)
using a Gemini XPS microplate spectrofluorometer from
Molecular Devices. The relative fluorescent unit (RFU) was
converted to concentration using a standard CoA curve.
varying concentrations of DMSO (0.2−3%). Enzyme activity
was also examined in the presence of Tween 20 and Triton
X-100 (0−4%) under similar conditions described above except
in the presence of 1% DMSO.
Molecular Mass Determination of OleD. The molecular
mass of His -tag S. maltophilia OleD was estimated by HPLC
on a BioSep-SEC-S4000 (300 mm × 7.8 mm, Phenomenex)
gel filtration column. The protein (100 μg) was eluted with
0 mM monobasic sodium phosphate (pH 7.2) and 100 mM
NaCl at a flow rate of 0.5 mL/min. β-Amylase (200 kDa),
alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic
anhydrase (29 kDa), and cytochrome C (12.4 kDa) were used as
molecular weight standards. The void and total column volume
were determined from the elution volume of blue dextran and
DNP-aspartate (299 Da). Elution of the proteins from the column
was monitored by the absorbance at 280 nm. A calibration curve
was generated by plotting the retention time against the log of the
native molecular mass for the protein standards.
Steady-State Kinetic Analysis of OleD. Steady-state
kinetic measurements were conducted under initial velocity condi-
tions by UV spectroscopy or fluorescence while having the
substrate or cofactor held at a constant concentration and varying
the other. Kinetic parameters were calculated from the average of
minimally 2 sets of triplicates with the Michaelis−Menten equa-
tion using the curve fitting software Kaleidagraph 4.0 (Synergy
Software, Reading, PA).
6
2
RESULTS
■
Sequence Alignment Analysis. A multiple sequence
alignment of putative OleD proteins from various organisms
was performed using Geneious 5.3 (see Supporting Information
Figure S1). The sequence identity among the proteins varied
from 40 to 70%. The alignment revealed the presence of the
conserved nucleotide binding motif (Rossman fold) in the
N-terminal region and catalytic triad SX YXXXK of the SDR
n
18,21,27−30
superfamily, in OleD.
The spatial orientation of the
Gly residues within nucleotide binding site, GXXGXXG, is
characteristic with the pattern displayed by the extended SDR
20,21,31
LC−MS Analysis of Reaction Products. Assays were per-
family.
Despite the low sequence identities (∼14−20%)
formed at 25 °C in the presence of 100 mM sodium phosphate
between OleD and SDR proteins, the nucleotide binding motif
and catalytic triad suggest a shared catalytic mechanism, which
is believed to be initiated by the transfer of a proton from the
Tyr residue to the carbonyl moiety on the substrate followed by
hydride transfer. The Ser facilitates catalysis by stabilizing the
substrate within the active site, whereas the Lys residue forms a
hydrogen bond with the ribose hydroxyl group of the cofactor
which in turn is hydrogen bonded to the Tyr residue. The
(
pH 7.5), 1 mM TCEP, 0.5% Tween 20, 0.2 mM (2R/S)-2-
hexyl-3-ketodecanoic acid (9) or syn-2-hexyl-3-hydroxydecanoic
acid (17) (in the presence of DMSO to give a final
concentration of 1%), 0.5 mM NADP(H), and 1 μM OleD.
Samples were taken at specific time points (0−60 min) and
analyzed by LC−MS in the negative mode on a Bruker
MicroTOF-Q equipped with an Agilent 1200 Series LC system
using a Discovery HS C18 (250 × 2.1, 5 μm, Supelco) column
at a flow rate of 0.3 mL/min. Buffer A was 5 mM ammonium
acetate in water and Buffer B was 5 mM ammonium acetate in
acetonitrile. The following solvent gradient was used to in the
analysis (time, % Buffer B): 0 min, 5%; 30 min, 100%; 35 min,
resulting proton relay system presumably lowers the pK of the
a
18,27
Tyr-OH to promote proton transfer.
The working hypothesis for OleD which was verified through
this biochemical study was that it would utilize 2-alkyl-3-
ketoalkanoic acid as a substrate and not the corresponding
thioester. β-Hydroxybutyrate dehydrogenase (HBDH) catalyzes
the reversible oxidation of 3-hydroxybutyrate to acetoacetate in
1
00%; 38 min, 5%; and 45, min 5%.
Assays involving OleA (5 μM) were performed at 25 °C in
the presence of 100 mM sodium phosphate (pH 7.5), 1 mM
+
the presence of NAD . Studies of this enzyme from
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a
Table 2. Steady-State Kinetic Parameters of S. maltophilia OleD
substrate
Km (μM)
kcat (min−1)
k /K (min−1 mM−1
)
cat
m
(
2R/S)-2-Hexyl-3-ketodecanoic acid (9)
161 ± 17
202 ± 17
181 ± 26
214 ± 23
31 ± 10
24 ± 2
58 ± 4
72 ± 3
79 ± 3
122 ± 3
85 ± 7
362 ± 21
b
syn-2-Hexyl-3-hydroxydecanoic acid (17)
2R,3S)-2-Hexyl-3-hydroxydecanoic acid (17a)
360 ± 33
435 ± 12
574 ± 57
2876 ± 740
(
b
syn-2-Octyl-3-hydroxydodecanoic acid (18)
syn-2-Decyl-3-hydroxytetradecanoic acid (19)
NADP+
b
c
d
NADPH
26 ± 4
a
b
Kinetic values were determined with at least three sets of triplicates to calculate the standard deviation. The syn compounds are composed of
c
(
2R,3S)- and (2S,3R)-enantiomeric pair. Kinetic parameters were obtained in the presence of saturating concentrations of syn-17 and determined by
d
UV-spectroscopy. Kinetic parameters were obtained in the presence of saturating concentrations of 9 and determined by fluorescence.
Pseudomonas putida have shown that a strictly conserved tetrad
subsequent analyses were carried out in the presence of 1%
DMSO and 0.5% Tween 20.
composed of a His, Lys, and two conserved Gln residues
32
coordinate the carboxylate group of 3-hydroxybutyrate. In the
human type 2 hydroxybutyrate dehydrogenase, three conserved
Arg residues, referred to as a “triple R” motif, are shown to be
Substrate Specificity and Steady-State Kinetic Anal-
yses. Through a nonstereospecific synthetic route, four
enantiomers of 2-alkyl-3-hydroxyalkanoic acid which could be
separated into diastereomers were generated. The syn-2-alkyl-3-
hydroxyalkanoic acids consist of the (2R,3S)- and (2S,3R)-
enantiomeric pair, whereas the anti-2-alkyl-3-hydroxyalkanoic
acids are composed of the (2S,3S)- and (2R,3R)-enantiomeric
pair. Subsequent work allowed for the stereochemical synthesis
of each isomer of 2-hexyl-3-hydroxydecanoic acid (17a, 17b,
33
coordinated to the carboxylate group of the substrate. There
are a number of highly conserved Arg, Asn, and His residues
found in OleD proteins (see Supporting Information Figure S1);
however, with the exception to Arg179 (based on SmOleD
numbering), none are conserved in positions of known
carboxylate coordinating residues found in P. putida and
human 3-hydroxybutyrate dehydrogenase proteins.
1
7c, and 17d). In addition, (2R/S)-hexyl-3-ketodecanoic acid
Protein Expression and Purification Molecular Mass
Determination. OleD from S. maltophilia and OleA from
K. radiotolerans were successfully overproduced in E. coli
(
9) and 3-ketodecanoic acid (27) were prepared via non-
stereospecific routes.
Each of the synthetic compounds, as shown in Table 1, were
screened by UV spectroscopy, fluorescence, and/or LC−MS as
potential substrates for purified OleD. In the oxidative direction
BL21(DE) as soluble N-terminal His -tag fusion proteins and
6
purified to greater than 95% purity by a single step nickel metal
affinity chromatography. Analysis of purified OleD by SDS−
polyacrylamide gel electrophoresis shows a single band with an
apparent molecular weight of ∼36 kDa, and when analyzed by
+
using NADP , the 2-alkyl-3-hydroxyalkanoic acids were
generally observed to be substrates and significantly higher
activity was seen in each case with the syn pair. Activity was
only observed with the free acids and not with the N-
MS, the recombinant His -tag protein had a mass of 36 472 Da,
6
consistent with the molecular mass calculated by the amino acid
sequence plus the mass of the N-terminal fusion. An apparent
acetylcysteamine (SNAC) thioester or methyl ester derivatives
+
(
Table 1). Similarly, no activity was observed using NAD . No
native molecular mass of 77 124 ± 1753 Da for the His -tag
6
activity was seen for (3R/S)-3-hydroxydecanoic acid (45) or
syn-2-butyl-3-hydroxyhexanoic acid (16). In the reductive
direction, (2R/S)-2-hexyl-3-ketodecanoic acid (9) was a sub-
strate, whereas 3-ketodecanoic acid (27) was not a substrate.
This preliminary data demonstrated that OleD catalyzes a
stereospecific reversible reaction with substrate specificity for
free acids and a C2-alkyl chain.
OleD was estimated by size-exclusion chromatography on a
calibrated BioSep-SEC-S4000 column, indicating that the
protein is a homodimer.
Effects of DMSO and Nonionic Detergents on OleD
Activity. Potential OleD substrates were generally poorly
water-soluble but readily soluble in DMSO. OleD assay condi-
tions with DMSO were therefore optimized using syn-2-hexyl-
A more detailed kinetic analyses of the OleD oxidative
reaction was made using syn isomers of 2-hexyl-3-hydrox-
ydecanoic acid (17), 2-octyl-3-hydroxydodecanoic acid (18),
and 2-decyl-3-hydroxydodecanoic acid (19) (Table 2). A
steady-state kinetic analysis with 17 and a fixed concentration
3
-hydroxydecanoic acid (17). Maximum relative activity was
observed at 0.5% and slowly decreased with increasing DMSO
see Supporting Information Table S1). Only a 6% decrease
(
from maximal activity was observed with 1% DMSO, and this
was selected for all assays to minimize any potential solubility
issues associated with longer chain substrates.
+
−1
of NADP provided an apparent k of 72 ± 3 min and a Km
cat
+
34
of 202 ± 17 μM. The analysis with variable NADP in the
Preparing fatty acids in a solution of cyclodextrin and/or
carrying out the assays in the presence of nonionic detergents,
such as Tween 20 or Triton X-100, are strategies that have
also been used to improve solubility of poorly water-soluble
presence of fixed concentration of 17 provided a Km of
2
4 ± 4 μM (Table 2). The catalytic efficiency increased with
increasing chain length with syn-2-decyl-3-hydroxytetradecanoic
acid (19) being the preferred substrate with a k /K 8- and 5-
35−41
compounds in an aqueous environment.
The effects of
cat
m
nonionic detergents and 1% DMSO on OleD activity are
summarized in Supporting Information Table S2. An increase
in enzyme activity with increasing Tween 20 concentration was
observed with maximum activity at 0.5%. A similar increase in
activity was observed when assayed in the presence of Triton
X-100; however, the relative activity was lower to that observed
in the presence of Tween 20. On the basis of these findings, all
fold higher than for the syn-2-hexyl-3-hydroxydecanoic acid
(17) and syn-2-ocyl-3-hydroxydodecanoic acid (18), respec-
tively (Table 2). Solubility issues precluded analysis of potential
substrates with longer alkyl chain lengths, such as 2-decyl-3-
hydroxyhexadecanoic acid (20). A steady-state kinetic analysis
was also carried out for the reductive reaction using a presumed
mixture of (2R/S)-2-hexyl-3-ketodecanoic acid (9), the 3-keto
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−1
analogue of 17, and provided the following: kcat 58 ± 4 min ,
Km 161 ± 18 μM (9) and 26 ± 4 μM (NADPH) (Table 2).
The syn isomers of the OleD substrates 17−19 comprise both
the (2R,3S)- and (2S,3R)-enantiomers. To determine the stereo-
specificity of the OleD reaction and in turn the stereochemical
course of the olefin biosynthetic pathway, a stereochemical
synthesis was used to generate each of the four isomers of
(2R/S)-hexyl-3-ketodecanoic acid (9) (elution time ∼29 min)
was incubated in the presence of OleD and NADPH. The new
peaks had a m/z of 271, consistent with the formation of
−
2-hexyl-3-hydroxydecanoic acid ([M − H] ) and coeluted with
synthetic syn- and anti-2-hexyl-3-hydroxydecanoic acid (17)
standard, respectively (data not shown). For the oxidative
reaction, the LC−MS analysis (Figure 3) revealed the
2-hexyl-3-hydroxydecanoic acid (17). As shown in Table 3, OleD
Table 3. Relative Activity of S. maltophilia OleD with 2-Hexyl-
3-hydroxydecanoic Acid Isomers
substrate
relative activity (%)
(2R,3S)-17a
(2S,3R)-17b
(2R,3R)-17c
(2S,3S)-17d
100.0
0.5
1.5
0.3
shows high stereoselectivity toward the oxidation of (2R,3S)-2-
hexyl-3-ketodecanoic acid (17a), with a greater than 98%
reduction for the other syn isomer (17b) or either anti isomer
Figure 3. TIC of OleD incubated in the presence of syn-2-hexyl-3-
hydroxydecanoic acid (17). Panel (A) represents time point 0 min and
(17c and 17d). Steady-state kinetic analysis with the enantiomer
(B) is the TIC of the reaction after 15 min. LC−MS analysis was
1
7a revealed comparable kcat and K values to those observed for
m
conducted in the negative mode.
the 17 syn pair (17a and 17b) (Table 2).
Confirmation of the OleD reaction products in both the
oxidative and reductive direction was achieved by LC−MS
analyses. As shown in Figure 2, two new peaks eluting at
conversion of syn-2-hexyl-3-hydroxydecanoic acid (17) (elution
time ∼30 min) to a peak with the retention time (∼29 min) and
−
−
MS properties (m/z 269, [M − H] ; m/z 225, [M − CO ] ) of
2
3
0 min (major) and 32 min (minor) were observed in the
(2R/S)-2-hexyl-3-ketodecanoic acid (9).
total ion chromatogram (TIC) when a presumed mixture of
OleA Activity and Product Identification. Our working
hypothesis for OleA was that it catalyzed condensation of two
acyl thioesters to generate a 2-alkyl-3-ketoacyl thioester. The
specificity of OleD for the 2-alkyl-3-ketoalkanoic acid indicated
either that OleA or another protein must first catalyze a hydro-
lytic step. A series of assays using the K. radiotolerans OleA
and the S. maltophilia OleD were carried out to address this
question, and to determine the overall stereochemical course of
the first steps of the olefin biosynthetic pathway.
OleA hydrolytic activity, as measured by CoA or N-acetyl-
cysteamine (SNAC) thioester release and fluorescence assay
involving ThioGlo-1, was examined in the presence of either
octanoyl-CoA, octanoyl-SNAC (44), or (2R/S)-2-hexyl-3-
ketodecanoyl-SNAC (10). In all cases, there was a time-
dependent release of free thiol. Octanoyl-CoA was the
preferred substrate with a 4- to 5-fold higher hydrolytic rate
compared to octanoyl-SNAC (44), and (2R/S)-2-hexyl-3-
ketodecanoyl-SNAC (10) (Table 4). Thioester release from
Table 4. Acyl-thioester Hydrolysis Rates of K. radiotolerans
a
OleA
substrate
rate(μM/min)
Octanoyl-CoA
Octanoyl-SNAC (44)
2R/S)-2-hexyl-3-kecodecanoyl-SNAC (10)
1.61
0.42
0.33
(
a
Rates were determined by fluorescence based assay using ThioGlo-1.
1
0 would provide 2-hexyl-3-ketodecanoic acid, an OleD
substrate. Consistent with this prediction, coincubation of 10+,
Figure 2. TIC of OleD incubated in the presence of (2R/S)-2-hexyl-3-
ketodecanoic acid (9). Panel (A) represents time point 0 min, (B) is
the TIC of the reaction after 15 min, (C) syn-2-hexyl-3-
hydroxydecanoic acid (17) synthetic standard, and (D) anti-2-hexyl-
NADPH, OleA, and OleD was shown to lead to NADP
generation, through an increase in fluorescence (see Supporting
Information Figure S2). This increase was not observed in
control experiments where the substrate, cofactor, or either
enzyme was absent.
3
-hydroxydecanoic acid (17) synthetic standard. LC−MS analysis was
conducted in the negative mode.
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Biochemistry
Article
A series of LC−MS experiments were used to verify that
OleA catalyzed both the condensation and hydrolytic steps and
that the OleA product was processed by OleD to provide the
syn isomer of 2-alky-3-hydroxyalkanoic acid. When OleA was
incubated with octanoyl-CoA, an ion with an m/z correspond-
conserved within the OleD proteins inferring NADP(H)
17,20,21,42
specificity.
The S. maltophilia OleD catalytic efficiency
was greatest with longer alkyl chain substrates, consistent with
the observation that saturated and unsaturated acyclic hydro-
carbons in the typical range C −C are made by S. maltophilia
22
31
−
ing to 2-hexyl-3-ketodecanoic acid (9) (m/z 269, [M − H] ;
and constitute approximately 0.25−2% of cellular dry
−
2,3,5−8,16
m/z 225, [M − CO ] ) eluting at ∼25 min was observed under
weight.
Shorter chain length alkenes are observed in
2
the conditions implemented (see Supporting Information
K. radiotolerans (C −C ) and the OleD from this organism
19
24
−
Figure S3). An ion with a m/z of 1018 ([M − H] )
might be expected to have different chain length specificity.
A sequence analysis revealed a 40% sequence identity and subtle
differences in hydrophobicity between OleD proteins from
S. maltophilia and K. radiotolerans. However, the analysis fails
to provide insight into the predicted differences in chain length
specificity for the two proteins. The enhancement of the
SmOleD catalytic efficiency with longer alkyl chains arose pre-
corresponding to 2-hexyl-3-ketodecanoyl-CoA eluting ∼20 min
+
was also observed. Upon addition of OleD and NADP , the ion
peak eluting at ∼25 min disappeared and a single new peak
−
emerged at ∼27 min, which had an m/z of 271 ([M − H] )
consistent with the formation of 2-hexyl-3-hydroxydecanoic
acid (17) (see Supporting Information Figure S4), and
coeluted with synthetic standard of syn-2-hexyl-3-hydroxydeca-
noic acid (17) (data not shown). Replacing octanoyl-CoA with
octanoyl-SNAC (44) thioester resulted in similar findings,
formation of 2-hexyl-3-ketodecanoic acid (9) with OleA alone
dominantly from lower K values, suggesting that the difference
m
arise mostly from stronger binding and not the subsequent
catalytic steps. The presence of the 2-alkyl chain is crucial for
enzyme recognition and catalysis as reflected by the lack of
OleD activity toward 3-ketodecanoic (27) and 3-hydroxydeca-
noic acids (45).
(
2
see Supporting Information Figure S5) and conversion to syn-
-hexyl-3-hydroxydecanoic acid (17) upon addition of OleD
and NADPH (see Supporting Information Figure S6). In this
The OleD reaction product in the reductive direction (Figure 1)
contains two stereogenic centers at C2 and C3. In principle, in
the olefin biosynthetic process, the stereochemical pattern of
the 2-alkyl chain is the direct consequence of the condensation
step catalyzed by OleA whereas the stereochemistry of the
3-hydroxy group is established by OleD. The highly stereo-
selective nature of OleD toward (2R,3S)-2-hexyl-3-hydroxyde-
canoic acid (17a) in the oxidative direction suggests the
stereochemistry of the 2-alkyl group as a result of OleA catalysis
is in the R-configuration and that OleD catalyzes the
stereospecific reduction of the 3-keto group to give a (3S)-
hydroxy group. Additional support for this stereochemical
assignment was the observation that a coincubation of OleA,
OleD, and NADPH and either octanoyl CoA or octanoyl-SNAC
(44) provided a syn isomer of 17 (which would be either 17a or
b). Conversely, when OleD was incubated with a (2R/S)-2-
hexyl-3-ketodecanoic acid (9), both syn and anti products of 17
were observed. Thus, the OleA reaction must provide a single
−
case, a peak with an m/z of 370 ([M − H] ) corresponding to
and having the same retention time (∼31.4 min) as 2-hexyl-3-
ketodecanoyl-SNAC thioester (10) was also observed (see
Supporting Information Figure S5). Assays with octanoyl-CoA
and octanoyl-SNAC were also conducted in the presence of
decanoic acid and resulted in the same findings. The absence of
any new products generated using the decanoic acid demon-
strated that both substrates for the initial reaction catalyzed by
OleA must be thioesters.
Incubating OleA in the presence of a presumed (2R/S)-2-
hexyl-3-ketodecanoyl-SNAC (10) resulted in the formation of
an ion that had an m/z and retention time (∼25 min)
consistent with 2-hexyl-3-ketodecanoic acid (9) (m/z 269,
−
−
[
M − H] ; m/z 225, [M − CO ] ) (see Supporting
2
Information Figure S7). In this case, however, the addition of
OleD resulted in the formation of two new compounds eluting
at 27 and 28 min, consistent with the formation of both syn-
−
and anti-2-hexyl-3-hydroxydecanoic acid (17) ([M − H] ) (see
2R enantiomer of 9 which is converted to 17a (the (2R,3S)-
Supporting Information Figure S8). The same observation of
syn and anti products was made using (2R/S)-2-hexyl-3-
ketodecanoic acid (9) with OleD and NADPH alone, and this
result suggests that both the 2R and 2S isomers of 10 are
substrates for OleA hydrolytic activity.
isomer) by OleD hydride addition to the re face at C3. Previous
preliminary deuterium labeling investigations of the olefin
pathway studies have demonstrated that the reductive step in
the process involves transfer of the 4-pro-R hydride from
10
NADPH to the substrate. At this point, the stereochemical
components of the OleD reaction have thus been elucidated.
The production of an anti isomer of 17 (17c or d) from racemic
mixture of 9 logically dictates that OleD can also process the 2S
isomer to generate an anti product, which must be 17d (the
(2S,3S)-isomer). In this case, the hydride addition occurs again
to the re face at C3. While the processing of a 2S isomer by
OleD does not appear to be physiologically relevant (this is not
the product generated by OleA), it does indicate potential use as
a biocatalyst having a tolerance not only for different alkyl chain
lengths, but also for the stereochemical configuration of the C2
alkyl substituent.
DISCUSSION
■
Four genes annotated as oleA, oleB, oleC, and oleD have now
been identified as being responsible for generation of olefins
from activated fatty acid thioesters in a number of organisms,
2−9
including S. maltophilia and K. radiotolerans.
Despite the
exciting potential for the olefin pathway in the generation of
biofuels and other specialty chemicals, most of the individual
biochemical steps catalyzed by these the ole gene products have
not been delineated experimentally.
This work has shown that OleD from S. maltophilia
(
2
SmOleD) catalyzes the reversible NADPH reduction of
-alkyl-3-ketoalkanoic acids. No activity is observed with
Our work on the K. radiotolerans OleA described herein and
recent independent work on the X. campestris OleA not only
9
NAD(H), which is consistent with the observation that the
conserved Asp residue found in all NAD(H) preferring enzymes
is absent in a comparable position in OleD proteins.
Furthermore, the presence of a basic Arg residue located ∼19
residues from the Gly-rich motif involved in cofactor binding is
demonstrates that they catalyze a condensation of two acyl
thioesters to generate a 2R-acyl-3-keto acyl thioester, but that
there is a subsequent hydrolysis step to generate the cor-
responding free acid. Two pieces of evidence suggest that the
condensation step occurs fast relative to the second hydrolysis
17,20,21,42,43
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dx.doi.org/10.1021/bi201096w|Biochemistry 2011, 50, 9633−9640

Biochemistry
Article
step: (1) using either the CoA or NAC thioesters, the inter-
mediate 2R-acyl-3-keto acyl thioester was observed in reactions
with OleA; and (2) similar hydrolytic rates are observed
between octanoyl-SNAC (44) and (2R/S)-2-hexyl-3-ketodeca-
noyl-SNAC (10). In the latter case, a faster rate of hydrolysis
with 10 would be expected if condensation were the slow step.
Together, these observations have elucidated the role and
stereochemical course of OleA and OleD in the first steps of
the olefin pathway, leading to a 2-alkyl-3-hydroxyalkanoic
acid pathway intermediate (Figure 1). Attaining the olefin from
this intermediate would require decarboxylation and dehy-
dration steps. These might occur separately or as a combined
decarboxylative−elimination step. There is strong genetic
(4) Albro, P. W., and Dittmer, J. C. (2002) Biochemistry of long-
chain, nonisoprenoid hydrocarbons. I. Characterization of the hydro-
carbons of Sarcina lutea and the isolation of possible intermediates of
biosynthesis. Biochemistry 8, 394−405.
(
5) Wackett, L. P. (2008) Microbial-based motor fuels: science and
technology. Microb. Biotechnol. 1, 211−225.
6) Tornabene, T. G., and Peterson, S. L. (1978) Pseudomonas
(
maltophilia: Identification of the hydrocarbons, glycerides, and
glycolipoproteins of cellular lipids. Can. J. Microbiol. 254, 525−532.
(7) Sukovich, D. J., Seffernick, J. L., Richman, J. E., Hunt, K. A.,
Gralnick, J. A., and Wackett, L. P. (2010) Structure, function, and
insights into the biosynthesis of a head-to-head hydrocarbon in
Shewanella oneidensis strain MR-1. Appl. Environ. Microbiol. 76, 3842−9.
(8) Beller, H. R., Goh, E.-B., and Keasling, J. D. (2010) Genes
involved in long-chain alkene biosynthesis in Micrococcus luteus. Appl.
Environ. Microbiol. 76, 1212−1223.
7,9,10,16
evidence for the role of the oleC gene in the olefin pathway,
and we hypothesize that it is involved in these final steps.
Biochemical investigations of OleC will be reported in due course.
(9) Frias, J. A., Richman, J. E., Erickson, J. S., and Wackett, L. P.
(2011) Purification and characterization of OleA from Xanthomonas
campestris and demonstration of a non-decarboxylative Claisen
condensation reaction. J. Biol. Chem. 286, 10930−10938.
ASSOCIATED CONTENT
Supporting Information
The detailed experimental procedures, characterization data of
all synthesized compounds, and the LC−MS extracted ion
chromatograms (EIC) of OleA reactions associated with this
article. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
*
S
(10) Friedman, L., Costa, B. D. (2008) Hydrocarbon-producing
genes and methods of their use, WIPO publication number WO 2008/
47781.
11) Albro, P. W. (1971) Confirmation of the identification of the
1
(
major C-29 hydrocarbons of Sarcina lutea. J. Bacteriol. 108, 213−218.
(12) Tornabene, T. G., Gelpi, E., and Oro, J. (1967) Identification of
fatty acids and aliphatic hydrocarbons in Sarcina lutea by gas
chromatography and combined gas chromatography-mass spectrom-
etry. J. Bacteriol. 94, 333−343.
AUTHOR INFORMATION
Corresponding Author
Office of Academic Affairs, Portland State University, P.O.
Box 751, Portland, Oregon 97207-0751, United States, Phone:
03-296-4829. Fax: 503-725-5262. E-mail: reynoldsk@pdx.edu.
■
*
(13) Tornabene, T. G., Bennett, E. O., and Oro, J. (1967) Fatty acid
and aliphatic hydrocarbon composition of Sarcina lutea grown in three
different media. J. Bacteriol. 94, 344−348.
5
(14) Tornabene, T., Morrison, S., and Kloos, W. (1970) Aliphatic
Author Contributions
These authors contributed equally to this work.
hydrocarbon contents of various members of the family Micro-
coccaceae. Lipids 5, 929−937.
‡
(15) Tornabene, T. G., and Markey, S. P. (1971) Characterization of
Funding
branched monounsaturated hydrocarbons of Sarcina lutea and Sarcina
flava. Lipids 3, 190−195.
This work was supported in part by the Portland State
University Miller Sustainability grant. Mass spectrometer was
carried out the Portland State Bioanalytical Mass Spectrometer
Facility which was supported in part by a Grant from the NSF
(16) Sukovich, D. J., Seffernick, J. L., Richman, J. E., Gralnick, J. A.,
and Wackett, L. P. (2010) Widespread head-to-head hydrocarbon
biosynthesis in bacteria and role of OleA. Appl. Environ. Microbiol. 76,
(
Grant no.: 0741993).
3
850−62.
(
17) Duax, W. L., Pletnev, V., Addlagatta, A., Bruenn, J., and Weeks,
ACKNOWLEDGMENTS
■
C. M. (2003) Rational proteomics I. Fingerprint identification and
cofactor specificity in the short-chain oxidoreductase (SCOR) enzyme
family. Proteins: Struct., Funct., Bioinf. 53, 931−943.
We thank Stephen del Cardayre, of LS9, Inc., for the initial
invitation to collaborate on this project, for the expression
plasmids, and for all the insightful discussions.
(18) Filling, C., Berndt, K. D., Benach, J., Knapp, S., Prozorovski, T.,
Nordling, E., Ladenstein, R., Jo
Critical residues for structure and catalysis in short-chain dehydro-
̈
rnvall, H., and Oppermann, U. (2002)
ABBREVIATIONS
■
genases/reductases. J. Biol. Chem. 277, 25677−25684.
SDR, short chain dehydrogenase; HBDH, 3-hydroxybutyrate
dehydrogenase; Tris-HCl, tris(hydroxymethyl)-aminomethane-
hydrochloric acid; SNAC, N-acetylcysteamine; TCEP, tris(2-
carboxyethyl)phosphine; ThioGlo-1, 10-(2,5-dihydro-2,5-
dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-methyl ester; EIC, ex-
tracted ion chromatogram; TIC, total ion chromatogram.
(
̈ ̈ ̈
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(
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(
2002) Short-chain dehydrogenases/reductases (SDRs). Eur. J.
Biochem. 269, 4409−4417.
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(
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