Exploring substrate scope and stereoselectivity of P450 peroxygenase OleTJE in olefin-forming oxidative decarboxylation

Article abstract of DOI:10.1039/c6cc04345c

As recently reported, the olefin-forming oxidative decarboxylation of straight-chain C₄-C₂₂ carboxylic acids catalyzed by P450 peroxygenase OleT_JE constitutes a mild alkene synthesis. The present study shows that structurally different carboxylic acids are also accepted, including those that generate di-substituted olefins. Exploratory mutational experiments lead to increased trans-selectivity.

Full text of DOI:10.1039/c6cc04345c

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decarboxylation of substrate
3
no subsequent isomerization binding pocket. Unfortunately, we discovered that the
expression level of OleTJE under the applied conditions is too
low for delivering enough protein in the usual 2-ml wells of 96-
format microtiter plates for reaction and screening to be
possible. Consequently, the conventional saturation
mutagenesis experiments at multiple CAST sites could not be
performed in the usual manner. Nevertheless, at this point we
decided to perform a few exploratory protein engineering
experiments, beginning with “rational” site-specific
occurs. Diastereoselectivity in the case of substrate
meager with slight preference for the trans-olefin (
3).
5
is
6 : 7 = 67 :
3
8
mutagenesis guided by the crystal structure of OleTJE. Since all
of the present substrates are fairly sterically demanding, we
speculated that space needs to be created for them to enter
and to bind more efficiently at the active site. Consequently,
two residues lining the entrance (Phe46 and Phe294) and
three CAST residues (Phe79, Phe173 and Phe291) were
considered for potential mutagenesis (Fig. 1a).
(
a)
F79
F173
Scheme 2. Substrates tested for OleTJE catalyzed olefin-
forming oxidative decarboxylation.
F46
F291
F294
Table 1. Wild-type OleTJE catalyzed olefin forming oxidative
decarboxylation.
L78
(
b)
Substrate
TON
Regioselectivity
Trans/cis-
selectivity
R245
1
1
1
1
1
3
5
8
1
a
b
c
d
e
428±10
215±10
38±3
64±5
251±10
14±2
72±5
196±10
86±5
-
-
-
-
-
-
-
-
-
-
-
-
I170
F173
F291
A249
F79
(6 + 7) : 4 = >99 : 1
9 : 10 = >99 : 1
-
6 : 7 = 67 : 33
-
-
P246
1
Conditions: Enzyme assays were performed in 2 mL glass vials
sealed with a PTFE (polytetrafluorethylene) septum. The whole
system volume is 500
CAMAB, 100 mM ammonium formate, 200
substrate, 5% DMSO worked as co-solvent, 100 mM phosphate
buffer, pH=7.4; incubation at 30 °C, 170 rpm for 24 h.
µ
L, 6
µ
M OleTJE, 2 U/mL FDH, 0.05 U/mL
M NAD+, 10 mM
µ
V292
L295
One of the astounding results is the observation that in all
cases only traces of undesired hydroxylation products are
formed. For example, the GC/MS analysis of the crude reaction
product in the conversion of 1a shows the presence of less
than 1-2% of hydroxylated product. This stands in contrast to
the known reactions of straight-chain carboxylic acids, which
generally result in appreciable amounts of undesired side-
products comprising hydroxylated compounds. Apparently
such side-reactions depend upon the structure of the starting
compound.
Figure 1. a) Five phenylalanine residues (purple) in OleTJE
considered for site specific mutagenesis in order to widen the
entrance (Phe46 and Phe294) and to provide more space at
the active site (Phe79, Phe173 and Phe291), based on the
crystal structure (PDB code: 4L40) harboring C20 fatty acid
8
black). The pink sphere symbolizes heme-Fe as catalytically
(
the active site. b) Docking of substrate 1a into the binding
pocket of WT OleTJE featuring H-bonding with Arg245 (blue
In order to increase enzyme activity, we considered
1
7-27
directed evolution
Saturation Test (CAST)
using the Combinatorial Active-Site dashed lines) and π/π-stacking of the substrate’s phenyl group
at selected sites lining the and Phe173 (orange dashed line).
1
9, 28, 29
2
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In order to test the above decision and to generate a Tyr, His, Asn, Asp, Cys, Arg, Ser, Gly).
COMMUNICATION
2
8, 29
In each case about
guide for further protein engineering, we docked substrate 1a 80-100 samples were picked and sequenced (see SI for details).
into the crystal structure of OleTJE (Fig. 1b). It reveals hydrogen All new mutants were then expressed separately in 50 ml
bond-based binding of its carboxyl function with Arg245 as bottles, which provided sufficient protein for activity
well as π/π stacking of its phenyl group with Phe173.
determination. The best variant proved to be F79L, but again
activity did not improve significantly, except in the reaction of
Table 2. Catalytic profile of OleTJE mutants as catalysts in
olefin-forming decarboxylation of carboxylic acids
substrate
reaction of substrate
= 79 : 21.
Finally, we were curious to see if the amide of carboxylic
5
(Table 2, lower part). Diastereoselectivity in the
.
5
in favor of the trans-olefin increased to
OleTJE
Substrate
TON
Regioselectivity
Trans/cis-
6 : 7
variants
selectivity
Phe294Ala
1a
469±10
256±10
40±2
-
-
acid 1a, compound 13, undergoes the analogous reaction with
formation of 2a (Scheme 3). If a cationic mechanism is
followed (Scheme 1), a possible product could also be the
lactam formed by intramolecular capture of the intermediate
carbocation by the amide function. In a very slow reaction,
olefin 2a was formed. Under the usual conditions it was not
possible to detect any small amounts of 14 that may have
been produced.
1
1
1
1
3
5
8
1
b
c
d
e
-
-
-
-
78±5
-
-
288±10
16±2
-
-
-
-
89±5
(6+7):4 ≧99:1
6:7=71:29
272±10
91±5
9:10 ≧99:1
-
-
-
-
-
-
-
1
-
-
-
-
-
-
Phe79Leu
1a
383±10
228±10
55±3
Scheme 3. OleTJE catalyzed oxidative olefin-forming reaction of
1
1
1
1
3
5
8
1
b
c
d
e
amide 13.
In summary, we have shown that WT P450 peroxygenase
OleTJE accepts a surprisingly broad range of structurally
different carboxylic acids. Branching at the α-position of the
carboxylic acid is tolerated by this mild procedure, leading for
the first time to di-substituted alkenes. The surprising
observation that only traces of side-products comprising
hydroxylated compounds are formed is of practical and
theoretical interest. Unfortunately, the expression rate of
OleTJE is so poor that conventional directed evolution could
not be applied for boosting activity. However, exploratory
mutational experiments provided a mutant that enhances
trans-selectivity and doubles activity in one case. In general,
however, the mutants induce marginal increases in activity.
Future research needs to focus on developing a better
expression system for OleTJE, while also exploring the behavior
of other substrates.
86±5
291±10
38±3
150±10
208±10
96±5
(6+7):4 ≧99:1
6:7=79:21
9:10 ≧99:1
-
-
1
-
Conditions as in Table 1 (in all cases less than 1% undesired
hydroxylation product).
On the basis of this analysis, five variants were generated
by sites specific mutagenesis, all featuring alanine with its
sterically smaller methyl sidechain: Phe46Ala, Phe79Ala,
Phe173Ala, Phe291Ala and Phe294Ala. These few mutants
were then screened for activity using all of the substrates. One
of the variants, Phe294Ala, responded positively, but the
degree of activity improvement proved to be meager in most
cases (Table 2, upper part). We also tested this mutant as
catalyst in the reaction of C18 and C20 fatty acids. The mutation
Phe294Ala exerted no notable effect, which means that OleTJE
activity was not reduced.
This work was supported by the Max-Planck-Society and
the LOEWE Research Cluster SynChemBio is gratefully
acknowledged. We thank Prof. Dr. D. B. Janssen
(Groningen/Netherlands) for providing the CAMAB plasmid.
Notes and references
1
2
J. Bacha and J. Kochi, Tetrahedron, 1968, 24, 2215-2226.
R. A. Sheldon and J. K. Kochi, Organic reactions, 1972, 19
We then re-considered CASTing, but in an unconventional
,
procedure due to the low expression rate of OleT . In a first
JE
2
79.
step, eight CAST residues (78, 79, 170, 246, 249, 291, 292 and
3
4
J. A. Miller, J. A. Nelson and M. P. Byrne, The Journal of
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