Old yellow enzyme-catalyzed dehydrogenation of saturated ketones

Article abstract of DOI:10.1002/adsc.201000862

Enzymes from extremophiles have always been of great interest for biotechnology because of their ruggedness against various stress factors. We have isolated, cloned, heterologously expressed and characterized a thermostable old yellow enzyme (OYE) from Geobacillus kaustophilus. In addition to the expected 'enone' reduction, GkOYE also catalyzes the reverse reaction, i.e., the desaturation of C-C bonds adjacent to a carbonyl to give the corresponding α,β-unsaturated ketone. The reaction proceeds at the expense of molecular oxygen without the need for a nicotinamide cofactor and represents an environmentally benign alternative to known chemical dehydrogenation methods.

Full text of DOI:10.1002/adsc.201000862

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DOI: 10.1002/adsc.201000862
Old Yellow Enzyme-Catalyzed Dehydrogenation of Saturated
Ketones
a
a
b
c
Matthias Schittmayer, Anton Glieder, Michael K. Uhl, Andreas Winkler,
a
d
d
c
Simone Zach, Jçrg H. Schrittwieser, Wolfgang Kroutil, Peter Macheroux,
Karl Gruber, Spiros Kambourakis, J. David Rozzell, and Margit Winkler *
b
e
e,f
a,
a
ACIB GmbH (Austrian Centre of Industrial Biotechnology), c/o Institute of Molecular Biotechnology, Graz University of
Technology, Petersgasse 14, 8010 Graz, Austria
Fax : (+43)-316-873-9302; phone: (+43)-316-873-9333; e-mail: margit.winkler@acib.at
ACIB GmbH, c/o Institute of Molecular Biosciences, University of Graz, Humboldtstraße 50, 8010 Graz, Austria
Institute of Biochemistry, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria
Institute of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
CODEXIS, Penobscot Drive 200, Redwood City, CA 94063, USA
Current address: Solidus Biosciences, 409 Illinois Street, Suite 2073, San Francisco, CA 94158, USA
b
c
d
e
f
Received: November 17, 2010; Published online: February 9, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201000862.
[
5]
Abstract: Enzymes from extremophiles have always
been of great interest for biotechnology because of
their ruggedness against various stress factors. We
have isolated, cloned, heterologously expressed and
characterized a thermostable old yellow enzyme
sponding silyl enol ethers has also been explored,
but often the yields and turnover numbers are very
limited. In general, the applicability of the existing
chemical methods is limited by chemo- and regiose-
lectivity as well as their low environmental compati-
bility.
(
OYE) from Geobacillus kaustophilus. In addition
to the expected ꢀenoneꢁ reduction, GkOYE also cat-
alyzes the reverse reaction, i.e., the desaturation of
CÀC bonds adjacent to a carbonyl to give the corre-
Enzymatic dehydrogenation of ketones would rep-
resent a valuable “green” alternative, but there are
only a few examples described so far, the first being
the enoate reductase-catalyzed dismutation reaction
of cyclohex-2-enone moieties leading to the corre-
sponding a,b-unsaturated ketone. The reaction pro-
ceeds at the expense of molecular oxygen without
the need for a nicotinamide cofactor and represents
an environmentally benign alternative to known
chemical dehydrogenation methods.
[
6]
sponding phenols and cyclohexanones. Similarly, the
aromatization to phenols was reported as catalytic
[
7]
promiscuity or side reaction. Massey et al. created
artificial old yellow enzyme (OYE) with increased
redox-potential (À50 mV) compared to the natural
enzyme (À207 mV). The apo-enzyme (lacking the
natural flavin-cofactor in the active site) was reconsti-
tuted with a chemically modified (synthetic) flavin co-
factor. With this artificial desaturase the dehydrogen-
ation of several saturated ketones was demonstrat-
Keywords: dehydrogenation; desaturation; enzy-
matic oxidation; old yellow enzyme; oxidoreduc-
ACHTUNGTRENNUNGt ases; thermophiles
[
8]
ed. Herein, we report the first enzymatic desatura-
a,b-Unsaturated ketones are versatile intermediates tion that is catalyzed by a readily available, natural
in organic synthesis and a number of strategies for thermophilic old yellow enzyme from Geobacillus
their production from the saturated keto analogues kaustophilus.
has been described. Traditional protocols often
While screening microorganisms for active pharma-
[
1]
employ toxic phenylselenium derivatives or strong ceutical ingredient-metabolizing activities, we found
[2]
oxidants such as SeO , DDQ, or periodic acid. More that testosterone 1a was readily converted to its 1,2-
2
recently, o-iodoxybenzoic acid (IBX) has been de- didehydro analogue boldenone 1b (Scheme 1) by the
scribed as a useful stoichiometric oxidant for the de- thermophilic microorganism Geobacillus kaustophilus
[
3]
saturation of carbonyl compounds. Pd-catalyzed de- DSM 7263. Boldenone was purified by preparative
[
4]
1
hydrogenation of saturated ketones or the corre- HPLC and identified by 2D- H NMR (see Supporting
268
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Adv. Synth. Catal. 2011, 353, 268 – 274

Old Yellow Enzyme-Catalyzed Dehydrogenation of Saturated Ketones
Scheme 1. Old yellow enzyme (GkOYE)-mediated testosterone 1,2-desaturation reaction.
Information). The responsible enzyme (Genbank Alternatively, highly pure enzyme was obtained by
YP 148185) was identified by MALDI-TOF after par- simple heat treatment of the lysate. Thereby, most E.
tial purification (see Supporting Information). It is a coli proteins were precipitated while the thermostable
homologue of the well characterized YqjM from Ba- GkOYE remained in the supernatant. As common
[
9]
[11]
cillus subtilis (66% amino acid sequence identity/ among OYEs,
0% similarity) and therefore belongs to the “old non-covalently bound, as shown by trichloroacetic
yellow enzyme” (OYE) family of flavoproteins. The acid precipitation. MS analysis of the supernatant re-
the active site prosthetic flavin is
8
[
10]
corresponding gene was then cloned and expressed vealed the active site flavin to be flavin mononucleo-
in E. coli at expression levels of up to 8 gL .
À1
tide (FMN, see Supporting Information), a finding
For further characterization, Geobacillus kaustophi- subsequently corroborated by the elucidation of the
lus OYE (GkOYE) was purified from E. coli lysates X-ray crystal structure of GkOYE (see also Figure 3).
by standard chromatographic purification procedures.
As expected for an enoate reductase, GkOYE ex-
hibited high reductase activity on typical substrates
such as cyclopentenone 2b and cyclohexenone 3b
(
Scheme 2) in the presence of NADPH. The reverse
reaction, i.e., the dehydrogenation of 3a to yield 3b, is
À1 [12]
known to be strongly endothermic (+109 kJmol ).
Since the dehydrogenation at the expense of molecu-
lar oxygen is not spontaneously happening and may
require high activation energy, we expected that the
reaction may be favoured at elevated temperatures
Scheme 2. Enone reduction and reverse reaction catalyzed only. Hence, the thermostability of the enzyme for
by GkOYE. this biocatalytic dehydrogenation reaction was ex-
Figure 1. Temperature dependence of desaturation of 3a under screening conditions (crude E. coli lysates, <1 mg total pro-
À1
tein mL , 24 h). Comparison of GkOYE (black) and YqjM (grey). For normalization of expression effects, enzyme amounts
which resulted in the same saturation activity (substrate 3b, 378C) were used.
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269

Matthias Schittmayer et al.
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Figure 2. Far UV CD-spectroscopic analysis of GkOYE (top) and YqjM (bottom). The original CD signals at 220 nm mea-
sured at 18C intervals together with a fitted sigmoidal curve are shown on the left. CD spectra measured at temperatures be-
tween 20 and 908C with an interval of 58C are shown on the right.
[13]
ploited. Using 3a as the model substrate we indeed (protein unfolding ~508C, see Figure 2) impedes its
found an increase in conversion with rising tempera- useful application for this transformation. However,
ture, reaching an optimum at 708C (Figure 1). Tem- due to the different inactivation behaviour, YqjM re-
peratures above 758C led to unfolding of the protein mained partially active at higher temperature, thereby
with concomitant release of the flavin cofactor allowing minimal conversions even at 508C.
(
Figure 2). While dynamic light scattering experi-
Testing other substrates, we were pleased to find
ments and FMN fluorescence measurements (see that ketones other than cyclohexanone were convert-
Supporting Information) demonstrated aggregation of ed even more readily by GkOYE at 70 8C (Table 1).
YqjM and only partial FMN release at elevated tem- The low conversion of the initially discovered oxida-
perature, GkOYE remained in tetrameric structure tion of testosterone 1a to boldenone 1b for example,
À1
and denatured at 76-828C with total release of FMN. was increased to 71% (GkOYE 20 mg mL , 24 h,
YqjM from Bacillus subtilis – although similar to 708C, 10 mM substrate). Lower substrate concentra-
GkOYE in active site structure (Figure 3) and exhibit- tion (5 mM) and prolonged incubation time (48 h) led
ing highly similar redox potential (À207Æ2 mV vs. to almost quantitative conversion for this substrate.
À208Æ3 mV) – proved less suitable for the ketone Cyclopentanone 2a, for example, led to 55% conver-
dehydrogenation. Traces of 3a were oxidized to 3b sion.
(
Figure 1), but the limited thermal stability of YqjM
270
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ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2011, 353, 268 – 274

Old Yellow Enzyme-Catalyzed Dehydrogenation of Saturated Ketones
Figure 3. A: Structural superposition of GkOYE and YqjM. Both monomer structures are shown in a cartoon representation
and are coloured according to their secondary structures. GkOYE: red (b-strands), gold (a-helices), green (loops); YqjM:
blue (b), grey (a), cyan (loops). B: Close-up view of the active centers of the aligned enzymes GkOYE (green; FMN cofac-
tor yellow) and YqjM (cyan; FMN cofactor orange). Active site residues in 10 ꢃ distance to the FMN-N5 are represented as
stick model.
Table 1. Conversion of ketones (10 mM) to the correspond-
À1
ing a,b-unsaturated compounds by GkOYE (20 mgmL ,
24 h, 708C).
Entry
Substrate
Product
Conversion [%]
[
a]
1
2
3
1a
2a
3a
1b
2b
3b
71[
b]
55
[
b,c]
5
[a]
[b]
[c]
Determined by HPLC-MS.
Determined by GC-MS.
3b is further oxidized to phenol.
In contrast to the enone reduction, no nicotinamide
cofactor was required for these oxidative transforma-
tions, suggesting that the catalytic cycle was closed via
[
14]
Figure 4. Selected spectra during the reoxidation of GkOYE
by molecular oxygen showing the reappearance of oxidized
FMN over a time span of 20 s.
aerobic reoxidation of FMNH2.
As a proof for
oxygen consumption, initial experiments were carried
out with oxygen limitation, which resulted in dramati-
cally reduced yields. Subsequently, stopped-flow ex-
periments revealed that molecular oxygen is indeed our experiments, both enantiomers were transformed
able to re-oxidize the reduced flavin mononucleotide at comparable rates (E=1.2), thus allowing theoreti-
(
FMN) of GkOYE (Figure 4).
cally full conversion (Scheme 3, Table 2). In a similar
To get a more detailed view of the perspectives of fashion, 2-methylcyclohexanone 5a was converted to
this enzyme, regio- and enantioselectivities were de- 2-methylcyclohex-2-enone 5b and dihydrocarvone 6a
termined using various substituted cyclic ketones as to carvone 6b. In contrast to 4a–6a, GkOYE dehydro-
substrates. Dehydrogenation of racemic 2-methylcy- genated 3-methylcyclohexanone 7a on the less hin-
clopentanone 4a occurred exclusively on the more dered side leading to 5-methylcyclohex-2-enone 7b
sterically demanding side of the ketone, thus 2-meth- with 23% ee. The prochiral substrate 4-methylcyclo-
ylcyclopent-2-enone 4b was formed with 56% conver- hexanone 8a was converted to 4-methylcyclohex-2-
sion (185 mM substrate, 708C, 24 h). In contrast to enone 8b and also in this case the stereopreference
[
1]
the reported observations with artificial OYE, in was low (30% ee). The tendency of the reaction prod-
Adv. Synth. Catal. 2011, 353, 268 – 274
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asc.wiley-vch.de
271

Matthias Schittmayer et al.
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ucts to undergo double desaturation and thus aroma-
tization proved to depend on the substitution pattern.
While no cresol by-product was observed in the dehy-
drogenation of 7a, the desaturated products 5b and 8b
were converted further to o-cresol and p-cresol, re-
spectively (Table 2 footnote). All reaction products
were identified by comparison of both their retention
times and mass spectra with those of authentic stand-
ards (see Supporting Information).
Since the enzyme requires molecular oxygen, and
the solubility of molecular oxygen in water decreases
with higher temperature, the dehydrogenation was
also tested in the presence of 1 bar molecular oxygen
instead of air. By using this set-up the conversion of
2-methylcyclopentanone was significantly increased
from 56% to 78% within 24 h.
The enzymatic transformation described here rep-
resents a proof of principle for an environmentally
[
15]
benign biocatalytic option
for the preparation of
a,b-unsaturated ketones starting from their corre-
sponding saturated keto analogues.
Concluding, the enzymatic desaturation catalyzed
by GkOYE opens new synthetic routes to a number
of a,b-unsaturated compounds. New ketosteroids
might thus be accessible. In contrast to known ketos-
[
16]
teroid dehydrogenases,
GkOYE does not rely on
expensive nicotinamide cofactors or artificial electron
acceptors, because the reoxidation of FMN is solely
O2 dependent. Unlike ketosteroid dehydrogenases,
GkOYEꢁs application is, however, not limited to ste-
roids, allowing the synthesis of small a,b-unsaturated
ketones, which are often valuable flavour and aroma
[
17]
defining compounds. Elevated temperatures appear
to be the key factor for the desaturase activity of old
yellow enzymes; whereas enzyme structure, redox po-
tential and several other physicochemical properties
of YqjM and GkOYE are highly similar, their thermal
behavior differs. GkOYE is available in high amounts
Scheme 3. Regioselective ketone dehydrogenation catalyzed
by GkOYE.
[8]
and is, in contrast to OYEs with artificial cofactor,
the first natural desaturase.
Table 2. Regioselective conversion of ketones to the corre-
sponding
a,b-unsaturated
compounds
by
GkOYE
À1
(5 mgmL , 24 h, 708C).
Experimental Section
[a]
[b]
[b]
[c]
No Subst. Prod. Conv. [%] eeS [%] eeP [%]
E
Typical Desaturation Reaction
1
2
3
4
5
4a
5a
6a
7a
8a
4b
5b
6b
7b
8b
56[
8
n.a.
n.a.
>99
23
1.2
2.6
n.a.
1.4
n.a.
d]
À1
35
20[
46
3
Protein solution (500 mL, 5–20 mgmL ) was added to a
10 mM substrate solution in 500 mL of a 50 mM KP buffer,
pH 7, and incubated at 708C for 24 h. All reactions were an-
alyzed by HPLC-MS, GC-FID or GC-MS using authentic
standards (where available) for comparison.
e]
29
16
i
[f]
6
n.a.
30
[
[
[
[
[
a]
Determined by GC-MS.
Determined by GC.
Enantioselectivity.
% of o-cresol formed as by-product.
Diastereomeric excess, commercial 5a is a mixture of
2R,5R) and (2S,5R)-isomers with a de of 57%.
% of p-cresol formed as by-product. n.a.: not applica-
ble.
b]
c]
d]
e]
CD Measurements
2
Freshly purified samples of GkOYE and YqjM were diluted
À1
(
6
to a concentration of 1 mgmL in 50 mM potassium phos-
[
f]
phate buffer pH 7.3. To clarify if FMN is set free before or
concomitant with protein unfolding, two independent tem-
272
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Adv. Synth. Catal. 2011, 353, 268 – 274

Old Yellow Enzyme-Catalyzed Dehydrogenation of Saturated Ketones
perature scans were performed on a Jasco J-715 spectropo- Acknowledgements
larimeter at the absorption maxima of FMN (373 nm) and
the protein (220 nm), respectively. These temperature scans
ranged from 208C up to 908C with a step size of one
degree. A 0.02 cm cuvette was used. In addition, a whole
wavelength scan (185–255 nm) was collected every 58C. For
the FMN measurements, the concentrations of the enzyme
samples were in the range of 4.5 mgmL and a 0.1 cm cuv-
ette was used. This relatively high concentration was neces-
sary to get a measurable CD signal.
We gratefully acknowledge Hannelore Mandl for excellent
technical support, Prof. Hansjçrg Weber (Institute of Organic
Chemistry, Graz University of Technology) for recording
NMR spectra, and the scientific staff at the EMBL/DESY
beamlines for their support during diffraction data collection.
We thank Codexis, the FFG, the Provinces of Styria, Vienna
and The Tyrol as well as the SFG for funding.
À1
Protein Crystallization
GkOYE was crystallized using sitting drop vapour diffusion References
yielding two different crystal forms (hexagonal and ortho-
rhombic). The structures were solved by molecular replace-
ment using a homology model based on the structure of
YqjM and were refined against 2.3 and 2.5 ꢃ datasets col-
lected on a rotating anode x-ray source as well as the
EMBL/DESY in Hamburg. Coordinates and structure fac-
tors were deposited in the Protein Data Bank (PDB) under
entry codes 3GR7 (hexagonal) and 3GR8 (orthorhombic).
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Adv. Synth. Catal. 2011, 353, 268 – 274