From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase

Article abstract of DOI:10.1002/anie.201605430

A new heme–thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position—a challenging reaction in organic chemistry—with H₂O₂as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC–MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of¹⁸O from the cosubstrate H₂¹⁸O₂, demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids.

Full text of DOI:10.1002/anie.201605430

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201605430
German Edition: DOI: 10.1002/ange.201605430
Enzyme Catalysis
From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal
Peroxygenase
Andrꢀs Olmedo, Carmen Aranda, Josꢀ C. del Rꢁo, Jan Kiebist, Katrin Scheibner,
Angel T. Martꢁnez, and Ana Gutiꢀrrez*
Abstract: A new heme–thiolate peroxidase catalyzes the
hydroxylation of n-alkanes at the terminal position—a chal-
lenging reaction in organic chemistry—with H2O2 as the only
cosubstrate. Besides the primary product, 1-dodecanol, the
conversion of dodecane yielded dodecanoic, 12-hydroxydode-
canoic, and 1,12-dodecanedioic acids, as identified by GC–MS.
Dodecanal could be detected only in trace amounts, and 1,12-
dodecanediol was not observed, thus suggesting that dodeca-
noic acid is the branch point between mono- and diterminal
hydroxylation. Simultaneously, oxygenation was observed at
other hydrocarbon chain positions (preferentially C2 and
C11). Similar results were observed in reactions of tetradecane.
The pattern of products formed, together with data on the
incorporation of 18O from the cosubstrate H218O2, demonstrate
that the enzyme acts as a peroxygenase that is able to catalyze
a cascade of mono- and diterminal oxidation reactions of long-
chain n-alkanes to give carboxylic acids.
cosubstrates and auxiliary enzymes, among other reasons,
applications of these versatile biocatalysts mainly focus on the
production of drug metabolites, pharmaceutical products, and
some specialty chemicals.[2,5,6]
A new heme peroxidase type was discovered 12 years ago
in the basidiomycete Agrocybe aegerita,[7] which efficiently
transfers oxygen to various organic substrates.[8,9] This
enzyme is able to catalyze reactions formerly assigned only
to P450s.[10] It differs from classical peroxidases by the
presence of a cysteine residue as the fifth ligand of the
heme iron atom,[11] and shares the heme–thiolate center with
P450s and with the chloroperoxidase from the ascomycete
Leptoxyphium fumago, which also has oxygenation activity.[8]
However, unlike P450s, which are intracellular enzymes,
whose activation often requires an auxiliary enzyme or
protein domain and a source of reducing power, the
A. aegerita enzyme is a secreted protein. It is therefore far
more stable, and more importantly only requires H2O2 for
activation.[8] In the latter sense, peroxygenase catalysis has
similarities with the so-called “peroxide shunt” operating in
P450s, and with a few P450s that show strictly peroxide-
dependent activity.[12] However, basidiomycete peroxyge-
nases generally have better catalytic and stability properties
than the above peroxide-activated P450s.
T
he selective oxyfunctionalization of saturated hydrocar-
bons under mild conditions is a major challenge in modern
chemistry. Among the thousands of reagents for organic
synthesis, few have been developed that are capable of the
selective oxidation of alkanes.[1] The alkane C H bond is
ꢀ
extremely inert and difficult to hydroxylate. Additionally, the
ꢀ
similarity of methylene C H bond strengths in a linear alkane
The A. aegerita peroxygenase was shown to catalyze
interesting oxygenation reactions on aromatic compounds,
and more recently its action on aliphatic compounds was
demonstrated,[13–16] thus expanding its biotechnological inter-
est. Therefore, the enzyme is known as an unspecific
peroxygenase (UPO). After the first peroxygenase from
A. aegerita (AaeUPO),[7] similar enzymes have been found in
other basidiomycetes, such as Coprinellus radians
(CraUPO)[17] and Marasmius rotula (MroUPO),[18] and
there are indications for their widespread occurrence in the
fungal kingdom.[19,20] Moreover, an UPO from the sequenced
genome of Coprinopsis cinerea (CciUPO) has been expressed
in an industrial host and shown to catalyze interesting
hydroxylation reactions.[15,21,22] UPOs could approach the
catalytic versatility of P450s and suitably supplement them in
the near future.[8] However, there are a number of reactions
that had not yet been shown for UPOs, including terminal
alkane hydroxylation.[8] Previous studies[13,14,22] showed the
hydroxylation of n-alkanes by AaeUPO and CciUPOs, but
the reaction is always subterminal (Figure 1).
and the lack of functional groups that can direct catalysis
make selective hydroxylation of these compounds highly
challenging. On the basis of their relative bond strengths, the
ꢀ
terminal methyl C H bonds are inherently more difficult to
oxidize than the secondary or tertiary C H bonds in the
ꢀ
hydrocarbon chain. Members of the cytochrome P450 mono-
oxygenase (P450) superfamily catalyze the selective oxy-
functionalization of many organic substrates under mild and
environmentally friendly conditions,[2] and some of them are
able to catalyze the terminal oxygenation of alkanes.[3,4]
However, owing to their frequent requirement for costly
[*] A. Olmedo, C. Aranda, Prof. J. C. d. . Rꢀo, Dr. A. Gutiꢁrrez
Instituto de Recursos Naturales y Agrobiologꢀa de Sevilla, CSIC
Reina Mercedes 10, 41012 Seville (Spain)
E-mail: anagu@irnase.csic.es
J. Kiebist, Prof. K. Scheibner
JenaBios GmbH
Orlaweg 2, 00743 Jena (Germany)
Prof. A. T. Martꢀnez
The recently described MroUPO presents differences
with respect to the most extensively studied UPOs, such as
higher activity towards aliphatic compounds, as well as the
ability to oxidize bulkier substrates,[8] and only shares
approximately 30% sequence identity. It was also known
Centro de Investigaciones Biolꢂgicas, CSIC
Ramiro de Maeztu 9, 28040 Madrid (Spain)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under http://dx.doi.org/10.
1002/anie.201605430.
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Figure 1. GC–MS analysis of the AaeUPO reaction with tetradecane,
showing the remaining substrate (Alk, alkane) and the subterminal
mono-/dihydroxylated (OH) derivatives (see the Supporting Informa-
tion for details).
that MroUPO presents differences in the active site, such as
a histidine residue (instead of a conserved arginine residue) as
ꢀ
a charge stabilizer for heterolytic cleavage of the H2O2 O O
bond (after transient proton transfer to a conserved glutamate
residue), thus resulting in compound I (CI) plus H2O,[8,11]
although their relevance in catalysis is still to be established.
Stimulated by these differences, we investigated the oxidation
of n-alkanes with this new UPO.
With this purpose, we tested two linear saturated long-
chain alkanes, n-dodecane and n-tetradecane, as MroUPO
substrates and identified the oxygenation products by GC–
MS. With a substrate concentration of 0.3 mm (in 20%
acetone), 68 and 45% conversion of dodecane and tetrade-
cane, respectively, was observed at 120 min in reactions with
MroUPO (0.5 mm). Under these conditions, the enzyme is
completely stable. The products of the reaction with dodecane
are shown in Figure 2A (see also Table S1 in the Supporting
Information), including those only formed by terminal
hydroxylation/s, such as 1-dodecanol, dodecanoic acid, w-
hydroxydodecanoic acid, and 1,12-dodecanedioic acid.
All the intermediates from an alkane 1 to a dicarboxylic
acid 10 via the terminal fatty alcohol 2 and w-hydroxy fatty
acid 5 (Figure 3, left) were identified in the MroUPO
reactions, except the monoaldehyde 3 (traces) and carboxy-
aldehyde 9, apparently as a result of their rapid further
oxidation. However, no terminal diol 6, w-hydroxyaldehyde 7,
or dialdehyde 8 (Figure 3, right) were observed. One explan-
ation is that conversion of the diol (if formed) into the diacid
is favored to such a degree that it proved impossible to
observe the aldehydes. Indeed, the rapid conversion of the
diol into the diacid was observed in the reaction of dodec-
anediol (Figure 2D) and tetradecanediol (not shown), and no
dialdehyde was observed. However, the possibility that the
diol 6 is not formed and the dicarboxylic acid is only produced
via the monocarboxylic acid 4 seems more feasible, since in
the reaction of dodecanol (Figure 2B) only dodecanoic acid
and its derivatives were identified. Indeed, the pattern of
products derived from dodecanol is similar to that for
dodecanoic acid (Figure 2C).
Figure 2. GC–MS analysis of MroUPO reactions with dodecane (A), 1-
dodecanol (B), dodecanoic acid (C), and 1,12-dodecanediol (D) show-
ing the remaining substrate (Alk, alkane; Alc, alcohol; and Ac, acid)
and the terminal (bold), terminal/subterminal (bold, italics), and
subterminal hydroxylated (OH) keto and carboxylic (COOH) deriva-
tives (see the Supporting Information for details).
ure 2A; see also Table S1). Therefore, in contrast with the
exclusively subterminal hydroxylation reactions of n-alkanes
by other UPOs (Figure 1), MroUPO is able to catalyze their
terminal hydroxylation (ca. 50% of products in Figure 2).
Moreover, a few products only showing subterminal oxygen-
ation were also identified as alkane hydroxy, keto, and
hydroxy/keto derivatives. When the alkane reactions were
performed at higher concentrations of acetone (40–60%) to
improve solubility, the proportion of the compounds formed
varied (see Table S1), probably as a result of increased
relative solubility of the substrates with respect to oxidized
intermediates. Finally, it was noted that higher conversion (up
Some of the terminal-oxygenation products showed addi-
tional oxygenation at subterminal (w-1 and w-2) positions,
with the formation of hydroxy and keto fatty acids (Fig-
2
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Figure 3. Pathways for the terminal oxygenation of n-alkanes to dicarb-
oxylic acids, including identified and hypothetical intermediates, and
three possible branch points (a, b and c) between mono- and
dioxygenated compounds.
to 100%) was observed at lower substrate concentrations (as
shown for 0.1 mm tetradecane in Figure S1 in the Supporting
Information).
The most characteristic property of UPO is its ability to
transfer oxygen to substrate molecules, which in the present
case includes a cascade of sequential mono- and diterminal
reactions of n-alkanes to give dicarboxylic acids. We therefore
investigated the origin of the oxygen atoms introduced into
the alkanes and intermediate compounds. The results of 18O
labeling reactions revealed that an oxygen atom from H218O2
(90% isotopic purity) is introduced into n-tetradecane to
form 1-tetradecanol, whose diagnostic fragment (m/z 271;
Figure 4A, top) appeared fully (90%) 18O-labeled (m/z 273;
Figure 4A, bottom). Direct evidence for the incorporation of
an oxygen atom from H218O2 in aldehyde formation could not
be obtained, since the aldehyde was barely detected. How-
ever, 18O was incorporated in the carboxyl group of myristic
acid, whose characteristic fragments (at m/z 285 and 117;
Figure 4B, top) became 18O-bilabeled (m/z 289 and 121;
Figure 4B, bottom). Likewise, H218O2 oxygen atoms were
incorporated into the fatty acid w-hydroxylated derivative
(see Figure S2A: 18O-trilabeled diagnostic fragments at m/
z 379 and 363) and dicarboxylic acid (see Figure S2B: 18O-
tetralabeled diagnostic fragment at m/z 395). In summary, the
reaction of tetradecane in the presence of H218O2 showed
18O labeling of the different hydroxy and carboxyl groups (see
the Supporting Information for details). Therefore, it can be
concluded that all oxygen atoms incorporated during alkane
oxidation by MroUPO are supplied by H2O2 and not from O2.
Figure 4. Mass spectra of 1-tetradecanol (A) and myristic acid (B)
from MroUPO reactions with n-tetradecane in 18O-labeling experiments
(bottom) and controls (top). The formulae for the unlabeled com-
pounds found in the H216O2 reactions (A and B, top) and the labeled
compounds found in the H218O2 (90% isotopic purity) reactions (A
and B, bottom) are shown as trimethylsilyl (TMS) derivatives.
The 18O-labeling results agree with the peroxygenation
mechanism depicted below,[8,9] whereby the resting enzyme
(RS), containing Fe3+ and a porphyrin (P), is activated by
H2O2 to yield CI, a Fe4+ O porphyrin cation radical (PC )
+
=
complex [Eq. (1)].
Cþ
P-Fe3þðRSÞ þ H2O2 ! P -Fe4þ¼OðC IÞ þ H2O
ð1Þ
ð2Þ
ð3Þ
Cþ
C
P
-Fe4þ¼OðC IÞ þ RH ! P-Fe4þ¼OðC IIÞ þ R
3þ
P-Fe4þ¼OðC IIÞ þ R ! P-Fe ðRSÞ þ ROH
C
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CI abstracts one H atom from the substrate (RH) to yield
AaeUPO used for comparison, and E. D. Babot (IRNAS) for
help in experimental assays.
a radical (RC) plus compound II (CII), a Fe4+ O reduced-
=
porphyrin complex [Eq. (2)]. Finally, CII completes de
ꢀ
hydroxylation reaction (R OH formation) and returns to
Keywords: alkanes · carboxylic acids · oxidoreductases ·
peroxygenase · terminal hydroxylation
RS [Eq. (3)]. The initial product of n-alkane oxidation by
MroUPO will be a terminal fatty alcohol, which is reported
herein for the first time for a peroxygenase reaction.[8,13] The
product of fatty-alcohol oxidation by the peroxygenase will be
a gem-diol from a second C1 hydroxylation, and will be either
i) directly hydroxylated (even at the nascent stage) to yield
a gem-triol intermediate, with irreversible dehydration to
release the fatty acid, or ii) first dehydrated to the aldehyde
and then hydroxylated to the fatty acid. Most 18O-labeling
data indicate that the gem-diol/aldehyde is immediately
hydroxylated (without hydroxyl exchange with the solvent);
however, the existence of minor simple labeling of the
carboxyl group in some MroUPO reactions (together with
double labeling) suggests some hydroxyl exchange with the
water at the aldehyde/gem-diol stage (only aldehyde traces
found in the chromatograms), although the loss of
18O labeling is much lower than reported for the P450 cascade
oxidation of hexadecanol.[3] Hydroxylation of the aldehyde
form was the mechanism suggested for the AaeUPO oxida-
tion of benzyl alcohol to benzoic acid, in which, in contrast
with our observations during this study, a substantial amount
of the aldehyde accumulated.[23] Finally, no diols or dialde-
hydes were detected in alkane oxidation by MroUPO, unlike
in the oxidation of n-hexadecane with P450.[3] Moreover, the
pattern of products identified in the alkane (and fatty-
alcohol) reactions suggests that diterminal oxyfunctionaliza-
tion by MroUPO initiates at the monocarboxylic acid
(Figure 3, reaction a) and not at the 1-alcohol or aldehyde
(Figure 3, reactions b and c, respectively).
Herein, we have described the first reaction cascade
leading to reactive carboxylic acids from chemically inert
alkanes with a peroxygenase. Having demonstrated the
feasibility of the enzymatic terminal oxyfunctionalization of
alkanes with MroUPO, we expect further studies to improve
the regioselectivity of the enzyme, whose structure has been
recently solved (PDB entry 5FUJ on hold), as reported for an
engineered P450 BM3 variant that shows approximately 50%
selectivity in the hydroxylation of the terminal position of
a medium-chain alkane.[24] This peroxygenase type has high
industrial potential for the mild activation of alkanes, with the
advantages of self-sufficient monooxygenase activity, thus
enabling large-scale transformations, and the ability to
hydroxylate the most unreactive terminal positions.
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Acknowledgements
This study was funded by the INDOX (KBBE-2013-7-613549)
EU project, and the BIORENZYMERY (AGL2014-53730-
R) and NOESIS (BIO2014-56388-R) projects of the Spanish
MINECO (cofinanced by FEDER). R. Ullrich and M.
Hofrichter (TU Dresden) are acknowledged for providing
Received: June 3, 2016
Revised: June 29, 2016
Published online: && &&, &&&&
4
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Communications
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Communications
Enzyme Catalysis
A. Olmedo, C. Aranda, J. C. d. . Rꢁo,
J. Kiebist, K. Scheibner, A. T. Martꢁnez,
A. Gutiꢂrrez*
&&&& — &&&&
From Alkanes to Carboxylic Acids:
Terminal Oxygenation by a Fungal
Peroxygenase
A peroxygenase from the fungus Maras-
mius rotula was found to catalyze a cas-
cade of mono- and diterminal oxygen-
ation reactions of long-chain n-alkanes to
carboxylic acids in the presence of H2O2
as the sole cosubstrate (see scheme).
This peroxygenase type has great advan-
tages for the mild activation of alkanes,
with its self-sufficient monooxygenase
activity and its ability to hydroxylate the
most unreactive terminal positions.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!