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unless otherwise stated. Enzyme assays were performed in potassi-
um phosphate buffer (100 mm, pH 7.2) containing KCl (100 mm)
and 10% glycerol. Aldehyde substrates were made up as stock sol-
utions in DMSO. Enzymatic reactions contained cAD (10 mm), fer-
rous ammonium sulfate (20 mm), PMS (75 mm) and NADH (1 mm),
C4–10 (2 mm) or C11–18 (300 mm) aldehyde in a total volume of
0.5 mL, respectively.
The decarbonylation reactions reported here were initially
performed in vitro. To demonstrate that long-chain fatty acids
are efficiently excluded from the A134F variant both in vitro
and in vivo, whole-cell biotransformations were performed
(Figures 5 and S2). From control experiments, small levels of
Gas chromatography detection of volatile hydrocarbon product
(C3–9 alkane) from enzyme reactions: Headspace analysis of vola-
tile products (C3–9 alkane) was carried out using GC. The reaction
mixtures were shaken continuously (190 rpm) at 378C. Headspace
samples (1.0 mL) were manually drawn off, and injected into the
GC with a syringe-lock needle at time intervals (0, 36, 72, 108, 144,
180 min for propane and 0, 5, 10, 20, 40, 60 min for n>3 alkanes).
All kinetic assays were performed in duplicate.
A Varian 3800 GC equipped with a DB-WAX column (30 mꢂ
0.32 mmꢂ0.25 mm film thickness, JW Scientific) was used to detect
and quantify the hydrocarbon released from enzyme reactions. The
column temperature was programmed as follows: 408C hold for
2 min, to 1008C at 208CminÀ1 (for detection of propane) and 408C
hold for 2 min, to 1508C at 208CminÀ1 (for detection of n>3 alka-
nes). The injector temperature was 2508C (10:1 split), and the FID
temperature was set at 2508C. The carrier gas was helium at a flow
rate of 1 mLminÀ1. Peak identification of each alkane was achieved
by comparison with pure alkane standards. Quantification of al-
kanes was achieved by comparison of integrated peak with calibra-
tion curves of standard pure alkanes.
Figure 5. All strains (E. coli control lacking cADO, E. coli transformed with
wild-type cADO or the A134F variant) were cultivated in lysogeny broth. All
reactions contained butanal (10 mm) and propane concentration was quanti-
fied from whole-cell biotransformations. (see the Supporting Information for
details).
propane were detected using an untransformed Escherichia
coli strain (0.03Æ0.01 mgLÀ1), the origin of which is unclear as
genome searches indicate this this organism does not contain
cADO-related genes. In broad agreement with in vitro turnover
data (Figure 4), the E. coli strain containing the A134F-variant
cADO generated propane at a rate (0.46Æ0.04 mgLÀ1) approxi-
mately twofold greater than E. coli containing wild-type cADO
(0.27Æ0.04 mgLÀ1). This elevation in activity is slightly less
than that determined from the in vitro turnover measure-
ments. We attribute this small difference to the approximately
twofold lower expression of the A134F variant in E. coli com-
pared with wild-type cADO (Figure S2).
Gas chromatography-mass spectrometry (GC-MS) detection of
less volatile hydrocarbon products (C10–17): Liquid-phase analysis
of nonvolatile products (C10–17 alkanes) was carried out using GC-
MS. At time intervals (0, 5, 10, 20, 40, 60, 80, 100 and 120 min), of
the reaction (1 mL) was terminated by extraction with ethyl acetate
(900 mL), containing an internal standard (0.005% limonene), and
dried over MgSO4. A 1 mL sample was then analysed using GC-MS
on a Varian 3800 GC instrument equipped with a Saturn 2000 ion
trap MS and a CP-8400 autosampler. A DB-WAX column (30 mꢂ
0.25 mmꢂ0.25 mm film thickness, JW Scientific) was used with the
following temperature programme: 708C hold for 2 min, to 2508C
at 208CminÀ1, hold for 2 min. The injector temperature was 2508C
(10:1 split), and the carrier gas was helium at a flow rate of
1 mLminÀ1. The transfer line, manifold and ion trap temperatures
were set to 250, 35 and 1508C, respectively. All kinetic assays were
performed in duplicate.
In summary, based on crystallographic data we have isolated
two variant forms of cADO that were strategically engineered
to display improved specificity for short- to medium-chain al-
dehydes. The A134F cADO has also been shown to generate
enhanced levels of propane production in whole-cell biotrans-
formations compared to wild-type cADO. These studies define
a region in the substrate channel that can be modified to
exclude longer-chain aldehydes and improve reactivity with
shorter-chain substrates. This simple switch in specificity releas-
es cADO from potential complications arising from long-chain
fatty acid binding in vivo. The A134F variant is an excellent cat-
alytic module from which to now explore the role of second-
shell residues to improve specificity and catalysis by cADO for
production of drop-in hydrocarbon biofuels.
Structural data have been deposited at RCSB Protein Data Bank
(IDs: 4KVQ, 4KVR and 4KVS)
Acknowledgements
This work was supported by grants from the European Union FP-
7 256808 to E.N.G.M. and N.S.S., and from NIH GM 093088 and
NSF CHE 1152055 to E.N.G.M. N.S.S. is a Royal Society Wolfson
Merit Awardee and holds an Engineering and Physical Sciences
Research Council (EPSRC) Established Career Fellowship. We
thank Diamond Light Source for access to beamlines I03
(MX1224-22) and I02 (MX7146-13), which contributed to the re-
sults presented here.
Experimental Section
Auxiliary PMS/NADH assay: All assays were performed under mi-
croaerobic conditions in an anaerobic glovebox (Belle Technology)
under a nitrogen atmosphere (oxygen maintained at <2 ppm)
Keywords: aldehyde
decarbonylation · enzyme catalysis · specificity
decarbonylase
·
biofuels
·
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 5
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