MhyADH catalysed Michael addition of water and in situ oxidation

Article abstract of DOI:10.1039/c0cc03229h

The Michael addition of water is a major challenge. Here an enzymatic approach is described. Interestingly, the enzyme (MhyADH) does not only catalyse the Michael addition of water but also the in situ oxidation of the product.

Full text of DOI:10.1039/c0cc03229h

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MhyADH catalysed Michael addition of water and in situ oxidationw
Jianfeng Jin,ab Philip C. Oskam,a Sanjib K. Karmee,a Adrie J. J. Straathofb and
Ulf Hanefeld*a
Received 13th August 2010, Accepted 9th September 2010
DOI: 10.1039/c0cc03229h
The Michael addition of water is a major challenge. Here an
enzymatic approach is described. Interestingly, the enzyme
(MhyADH) does not only catalyse the Michael addition of
water but also the in situ oxidation of the product.
described indirect assay for the Michael hydratase activity.10
In this coupled assay 3 is utilised as a substrate and compound
4 once generated is oxidised in situ to 5. The oxidation
is catalysed by an alcohol dehydrogenase also present in
A. denitrificans DSMZ 14773 (Scheme 1). Methylene blue or
dichlorophenol indophenol (DCPIP) as electron acceptor
allows this oxidation of 4 to 5 to occur while its reduction
can be followed UV-spectroscopically. The anaerobically
grown A. denitrificans DSMZ 14773 displayed higher activity
in this assay and from here on anaerobic growth conditions
were applied.z
The b-hydroxy carbonyl connectivity is widely spread in
nature, since it is part of the fatty acid metabolism and the
polyketide biosynthesis.1–3 Synthetic approaches are normally
based on Claisen-condensations or Aldol-type chemistry—
both in nature and in the laboratory.4,5 The Michael addition
of water to conjugated carbonyl compounds is little known.6–8
Only one enantioselective Michael addition of water has been
described, albeit indirect, proceeding via a hydroboration.9
This is surprising, all the more so since it would enable an
entirely new route towards b-hydroxy carbonyl compounds.9
In our search for an enzyme that would catalyse the Michael
addition of water to a,b-unsaturated carbonyl compounds and
more specifically a,b-unsaturated ketones such as 3, it was
noted that Alicycliphilus denitrificans DSMZ 14773 can
anaerobically degrade cyclohexanol (1).10–12 In the degrada-
tion pathway a Michael hydratase was postulated and indirect
evidence for its existence was presented (Scheme 1).10 Here we
describe the enrichment and catalytic activity of this unique
enzyme. To date only very limited knowledge about the
equivalent chemical reaction is available.6–9
To confirm the presence of the Michael hydratase it was
essential to develop analytical tools that would allow us to
follow the formation of 4 directly. The racemic reference
compound 4 was obtained selectively by a straightforward
cobalt-catalysed oxidation of 1,3-cyclohexanediol.13 With this
in hand a GC method for analysing the reaction was
established. However, the extremely high solubility of 4 in
water and its volatility made it difficult to isolate 4 or to
derivatise it for analysis.
As a direct test for the existence of the Michael hydratase,
cell free extract of A. denitrificans DSMZ 14773 was incubated
with 3. Extensive extraction and GC analysis revealed the
conversion of 3 to 4. This was further validated by GC-MS
and by co-injection of the extract with a synthetic sample of 4.
The yield of 4 reached approximately 15% after 1.5 h and no
further conversion was observed (Fig. 1).
A. denitrificans DSMZ 14773 was grown, both aerobically
and anaerobically, with 1 as the sole carbon source. Cell
extracts from both cultures were submitted to the earlier
To enhance the search for the Michael hydratase via the
coupled assay we first purified 3-hydroxycyclohexanone
dehydrogenase. To our surprise methylene blue or DCPIP
was still reduced when 3 was added to this purified alcohol
dehydrogenase. This result indicates that the hydration of 3
and the oxidation of 4 are catalysed by the same enzyme.
Scheme 1 Anaerobic degradation of cyclohexanol by Alicycliphilus
denitrificans DSMZ 14773.
a Gebouw voor Scheikunde, Afdeling Biotechnologie, Technische
Universiteit Delft, Julianalaan 136, 2628 BL Delft, The Netherlands.
E-mail: u.hanefeld@tudelft.nl; Fax: +31 (0)15-2781415
b Bioseparation Technology, Afdeling Biotechnologie, Technische
Universiteit Delft, Julianalaan 67, 2628 BC Delft, The Netherlands.
E-mail: A.J.J.Straathof@tudelft.nl
Fig. 1 The conversion of 2-cyclohexenone (3) to 3-hydroxy-cyclo-
hexanone (4) by MhyADH without the addition of an oxidising reagent.
w This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
c
8588 Chem. Commun., 2010, 46, 8588–8590
This journal is The Royal Society of Chemistry 2010

Fig. 3 The activity of MhyADH towards (3R)-4 (K) and racemic 4 (’).
Given the high water-solubility and volatility of 4 it proved to
be impossible to derivatise the analytical quantities of 4
obtained from the MhyADH catalysed addition of water to 3.
The substrate specificity of MhyADH was investigated by
screening a variety of a,b-unsaturated carbonyl compounds.
Both aldehydes and ketones proved to be substrates for
MhyADH (Table 1), although cyclohexenone 3 clearly was
the best substrate. No activity towards a,b-unsaturated
carboxylic acids was observed.
Fig. 2 The activity staining of MhyADH in the native protein gel.
MhyADH stained with methylene blue (60 mM), nitro blue tetrazolium
chloride (0.3 M), 2-cyclohexenone 3 (1 mM) (lane 1) or 3-hydroxy-
cyclo-hexanone 4 (1 mM) (lane 2). Lane 3 are protein markers:
albumin (66 000), lactate dehydrogenase (140 000), catalase (232 000),
ferritin (440 000), thyroglobulin (669 000).
Enriching these activities 43 times by a two-step purification
(DEAE Sepharose and Mono Q) yielded a single protein band
at B200 kD which did indeed contain both activities (Fig. 2).
This bifunctional enzyme first adds water to the CQC bond of
3 (Michael hydratase activity), resulting in 4 and then oxidises
this hydration product to 5 (alcohol dehydrogenase activity).
We therefore abbreviate it as MhyADH. Again, without
added oxidation reagent, MhyADH catalysed only the
Michael addition of water as described above (Fig. 1); how-
ever the enzyme was not stable under reaction conditions and
deactivation was observed. When methylene blue or DCPIP
was added, MhyADH catalysed both reactions (Scheme 2).
To probe the enantioselectivity of MhyADH enantiopure
(3R)-4 was prepared via kinetic resolution of rac-4 by Porcine
pancreas lipase-catalysed acylation in freshly distilled vinyl
acetate. The (3R)-acetate (ee = 92%) was deprotected by
ethanolysis catalysed by CALB (Novozym 435) in dry MTBE
yielding (3R)-4. The absolute stereochemistry of (3R)-4 was
determined by converting it into the known TBDMS ether.9,14
MhyADH was incubated with rac-4 and (3R)-4. While the
3R-alcohol was rapidly oxidised the racemic mixture was
converted much more sluggishly (Fig. 3). Indeed, it can be
clearly seen that the oxidation is inhibited by the S-enantiomer.
MhyADH thus displays R-selectivity in the oxidation step.
It has been reported that in the modular biosynthesis of
fatty acids, polyketides and nonribosomal peptides, large
multifunctional enzymes such as fatty acid synthase (FAS),
polyketide synthase (PKS) and nonribosomal peptide
synthetase (NRPS) are required to produce such primary
and secondary metabolites.15 These enzymes contain discrete
catalytic domains which are responsible for each step of the
biosynthesis and together they produce the corresponding
natural product. The bifunctionality of the MhyADH
described here might therefore be less uncommon in nature
Table 1 The substrate specificity of MhyADH as determined with the
coupled assayz
Substrate
Relative activity (%)
100
60.0
28.1
56.3
34.4
10.6
Scheme 2 MhyADH catalyses the Michael addition of water and the
subsequent enantioselective oxidation of the alcohol. In the absence of
an oxidation reagent the Michael product might be isolated.
For experimental detail see footnote z.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8588–8590 8589

than one may assume at first glance. The functions it
combines, however are exceptional. An enzyme that effectively
catalyses the unusual hydration of cyclic and acyclic
a,b-unsaturated ketones and aldehydes, i.e. the Michael
addition was to date unknown.16 Its ability to enantio-
selectively oxidise (in the presence of electron acceptors) the
Michael products only expands the applicability of this
enzyme. To summarise, because of its bifunctional nature,
MhyADH opens up a new route to 3-hydroxy carbonyl and
1,3-dicarbonyl compounds, both essential 1,3-difunctional
compounds.
concentrated using Centrion YM30 filter devices. The purified protein
was stored at ꢀ20 1C. The protein content was determined using a
Pierces BCA protein assay kit (Pierce Biotechnology).
Analytical methods: GC analysis of the reactions were performed
with a Shimadzu GC 2014 equipped with a CP-Wax 52 CB column
(2.0 mm ꢂ 50 m ꢂ 0.5 mm) using N2 as the carrier gas. The retention
time of 3 and 4 are 28.5 min and 36.6 min, respectively. Mass spectra
were determined with a Shimadzu GC-2010 Gas Chromatograph
coupled to a Shimadzu GCMS-QP2010S Gas Chromatographic Mass
Spectrometer.
The hydration reaction was performed in 10 mL of Tris–HCl buffer
(100 mM, pH 7.8) containing 0.1 mmol 2-cyclohexenone (3) and
3.1 U MhyADH activity. 0.5 mL samples were taken from the reaction
mixture every 30 min and extracted with 1.0 mL of ethyl acetate.
The coupled activity assay of 2-cyclohexenone hydratase and
3-hydroxycyclohexanone dehydrogenase (MhyADH) was performed
at room temperature in a quartz cuvette containing 0.9 mL reaction
mixture containing 1 mM substrate and 60 mM methylene blue or
DCPIP in 100 mM Tris–HCl (pH 7.8). The reaction was initiated by
adding 0.1 mL cell extract or enzyme solution. The spectra of the
reaction mixture were recorded on a Hewlett-Packard 8452A diode
array spectrophotometer at an interval of 2 s and absorbance change
at 578 nm (methylene blue, e = 20.7 cmꢀ1 molꢀ1; DCPIP, e =
16.8 cmꢀ1 molꢀ1) was determined.10 One unit is defined as the amount
of enzyme that reduces 1 mmol of DCPIP at pH 7.8 and 25 1C in one
minute.
Financial support for part of this project by B-Basic is
gratefully acknowledged. Permission to publish has been given
for the results obtained with B-Basic support.
Notes and references
z Chemicals were purchased from Sigma-Aldrich; 2-cyclohexenone,
1,3-cyclohexanediol, 2-buten-2-one, 3-penten-2-one, cinnamaldehyde,
2-hexenal and 2-octenal were distilled prior to use. A. denitrificans
DSMZ 14773 was purchased from DSMZ (Germany).
Cultivation of A. denitrificans DSMZ 14773 and enzyme purification:
the cells were cultivated in LB medium and minimal medium containing
cyclohexanol (1, 100 mg mLꢀ1). The minimal medium used for aerobic
cultivation contained (1 L distilled water): Na2HPO4ꢁ2H2O 3.5 g,
KH2PO4 1.0 g, (NH4)2SO4 0.5 g, MgCl2ꢁ6H2O 0.1 g, Ca(NO3)2ꢁ
4H2O 0.05 g, 1.0 mL trace element solution SL-4. The trace element
solution SL-4 contained (1 L distilled water): EDTA 0.5 g, FeSO4ꢁ
7H2O 0.2 g, trace element solution SL-6 100 mL.10 The trace element
solution SL-6 contained (1 L distilled water): ZnSO4ꢁ7H2O 0.10 g,
MnCl2ꢁ4H2O 0.03 g, H3BO3 0.30 g, CoCl2ꢁ6H2O 0.20 g, CuCl2ꢁ2H2O
0.01 g, NiCl2ꢁ6H2O 0.02 g, Na2MoO4ꢁ2H2O 0.03 g. The final pH was
7.2–7.4.10
The minimal medium used for anaerobic cultivation contained
(1 L distilled water): KH2PO4 0.816 g, K2HPO4 5.92 g, NH4Cl 0.53 g,
MgSO4ꢁ7H2O 0.2 g, KNO3 2.0 g, CaSO4ꢁ7H2O 0.025 g, cyclohexanol
(1) 0.1 g, 10 mL trace element solution SL-10 and 5 mL vitamin
solution.10 The final pH was adjusted to 7.2. The cultures were grown
on a rotary shaker at 180 rpm and 30 1C.
For the preparation of cell extracts, 1.5 g cells (wet weight) were
suspended in 10 mL Tris–HCl buffer (100 mM, pH 7.8). Cells were
disrupted by passage through a cooled French pressure cell with a
pressure difference of 139 MPa. Unbroken cells and debris were
removed by centrifugation at 100 000 ꢂ g for 1 h. The cell free extract
of A. denitrificans was applied to a 28 mL DEAE Sepharose column
previously equilibrated with buffer A (20 mM Tris–HCl, pH 7.8). The
elution was performed with buffer B (20 mM Tris–HCl, 1 M NaCl,
pH 7.8) from 0–50% at a flow rate of 5 mL minꢀ1. Fractions showing
MhyADH activity were pooled and concentrated using Centricon
YM30 centrifugal filter device (Millipore) in a centrifuge at 3000 ꢂ g.
After desalting with a PD-10 column, pooled fractions containing
MhyADH activity were applied to a MonoQ 5/50 GL column (1 mL)
(GE Healthcare) equilibrated with buffer A. The bound MhyADH
activity was eluted by a linear gradient from 0–50% of buffer B
at a flow rate of 0.4 mL minꢀ1. Active fractions were pooled and
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