Fatty Acid Chain Shortening by a Fungal Peroxygenase

Article abstract of DOI:10.1002/chem.201704773

A recently discovered peroxygenase from the fungus Marasmius rotula (MroUPO) is able to catalyze the progressive one-carbon shortening of medium and long-chain mono- and dicarboxylic acids by itself alone, in the presence of H₂O₂. The mechanism, analyzed using H₂¹⁸O₂, starts with an α-oxidation catalyzed by MroUPO generating an α-hydroxy acid, which is further oxidized by the enzyme to a reactive α-keto intermediate whose decarboxylation yields the one-carbon shorter fatty acid. Compared with the previously characterized peroxygenase of Agrocybe aegerita, a wider heme access channel, enabling fatty acid positioning with the carboxylic end near the heme cofactor (as seen in one of the crystal structures available) could be at the origin of the unique ability of MroUPO shortening carboxylic acid chains.

Full text of DOI:10.1002/chem.201704773

A Journal of
Accepted Article
Title: Fatty Acid Chain Shortening by a Fungal Peroxygenase
Authors: Andrés Olmedo, José C. del Río, Jan Kiebist, René Ullrich,
Martin Hofrichter, Katrin Scheibner, Angel T. Martínez, and
Ana Gutiérrez
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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To be cited as: Chem. Eur. J. 10.1002/chem.201704773
Link to VoR: http://dx.doi.org/10.1002/chem.201704773
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Fatty Acid Chain Shortening by a Fungal Peroxygenase
Andrés Olmedo, José C. del Río, Jan Kiebist, René Ullrich, Martin Hofrichter, Katrin Scheibner, Angel T.
Martínez, and Ana Gutiérrez*
Abstract: A recently discovered peroxygenase from the fungus
Marasmius rotula (MroUPO) is able to catalyze the progressive one-
carbon shortening of medium and long-chain mono- and dicarboxylic
acids by itself alone, in the presence of H2O2. The mechanism,
analyzed using H218O2, starts with an α-oxidation catalyzed by
MroUPO generating an α-hydroxy acid, which is further oxidized by
the enzyme to a reactive α-keto intermediate whose decarboxylation
yields the one-carbon shorter fatty acid. Compared with the
previously characterized peroxygenase of Agrocybe aegerita, a
wider heme access channel, enabling fatty acid positioning with the
carboxylic end near the heme cofactor (as seen in one of the crystal
structures available) could be at the origin of the unique ability of
MroUPO shortening carboxylic acid chains.
databases.[1;11] The peroxygenase from M. rotula (MroUPO)
shows several special features compared to other UPOs, for
example, the inability to oxidize halides, less pronounced
capacity to oxygenate aromatics,[10] and the unique ability for
terminal hydroxylation of n-alkanes.[12]
Despite the widespread occurrence of peroxygenases and
related heme-thiolate peroxidases, only two molecular structures
have been reported to date, corresponding to the classic
ascomycete chloroperoxidase (CPO),[13] and AaeUPO.[14]
Fortunately, the crystal structure of MroUPO has just been made
available (PDB entries 5FUJ and 5FUK). Interestingly, although
MroUPO is a dimeric protein due to an inter-monomer disulfide
bond (Figure 1B – while AaeUPO and CPO are monomers), this
fact does not affect the accessibility to the heme channel and
cofactor (Figure 1A).
The use of biocatalysts for organic synthesis replacing traditional
metal catalysts has several advantages, such as better regio-
and stereoselectivity, fewer side products, and potentially lower
environmental impact. Enzymes that catalyze the transfer of an
oxygen atom from peroxide to substrates are classified as
peroxygenases (EC.1.11.2). Unspecific peroxygenase (UPO, EC
1.11.2.1) is the most prominent member of this group because
of its versatility for oxygen transfer reactions[1] that makes it
highly attractive as industrial biocatalyst.[2-4]
A
The first UPO was described in the basidiomycetous fungus
Agrocybe aegerita (AaeUPO)[5] catalyzing reactions formerly
assigned only to P450 monooxygenases (P450s). However,
unlike P450s that are intracellular enzymes and often require a
flavin-containing auxiliary enzyme or protein domain and a
source of reducing power [NAD(P)H], UPO is a secreted protein,
therefore far more stable, and only requires H2O2 for activation.[2]
AaeUPO was shown to catalyze oxygenation reactions on
aromatic compounds,[6] and later, the action on aliphatic
B
compounds
was
demonstrated,[7;8]
expanding
its
biotechnological relevance.
Then, similar UPO proteins have been purified from other
basidiomycetes and ascomycetes such as Coprinellus radians,[9]
Marasmius rotula[10] and Chaetomium globosum;[11] and almost
three-thousand related sequences (from sequenced genomes
and environmental samples) are currently available in
Figure 1. Dimeric MroUPO. A) Solvent-access surface with one of the heme
cofactors (red spheres) visible through an access channel. B) Ribbon-like
model including the C227-C'227 disulfide bond (CPK-colored spheres), the
two cofactors (red and CPK sticks), and surface glycosylation (CPK sticks).
From 5FUJ.
[*] A. Olmedo, Prof. J. C. del Río, Dr. A. Gutiérrez
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC
Reina Mercedes 10, 41012 Seville (Spain)
E-mail: anagu@irnase.csic.es
Here, the one-carbon shortening of carboxylic acids, a
fascinating reaction catalyzed by MroUPO, is revealed. This
Jan Kiebist, Prof. Katrin Scheibner
JenaBios GmbH
Löbstedter Str. 80, 07749 Jena (Germany)
reaction was first evidenced when
a
dicarboxylic acid
René Ullrich, Prof. Martin Hofrichter
TU Dresden
Markt 23, 02763 Zittau, Germany
Prof. Angel T. Martínez
Centro de Investigaciones Biológicas, CSIC
Ramiro de Maeztu 9, 28040 Madrid (Spain)
Supporting information for this article is given via a link at the end of the
document.
(tetradecanedioic acid) was tested as substrate of the enzyme,
and the products were analyzed by gas chromatography-mass
spectrometry (GC-MS) (Figures 2A, 2B, S1 and S2). High (93%)
substrate (0.1 mM) conversion by MroUPO (~2 µM) was attained
in 24 h, yielding a series of chain-shortened dicarboxylic acids,
such as tridecanedioic (63% of products) and dodecanedioic
(13%) acids, together with α-, β- and γ-hydroxyderivatives. The
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10.1002/chem.201704773
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reaction was also studied with AaeUPO (Figure 2C), which failed
to convert the substrate.
oxidation (although, under some forced reaction conditions,
some two-carbon shortenings of β-hydroxy acid could be
observed as well; Fig. S4).
di-C14
(-1)-keto-C14
A
OH-di-C14
di-C14
A
α
β
α-enol-di-C14
di-C13
α-OH-C14
γ
di-C13
C14
18
20
20
20
26
26
26
22
24
C13
Retention time (min)
12
14
16
18
20
22
24
26
di-C13
Retention time (min)
B
di-C10
(-1)-keto-C10
B
C9
OH-di-C13
OH-di-C14
di-C12
α
β
di-C14
di-C11
di-C10
γ
di-C9
β
α
C10
γ
C8
α-OH-C9
18
22
24
8
10
12
14
16
18
20
22
Retention time (min)
Retention time (min)
di-C14
C
Figure 3. Comparison of tetradecanoic acid (A) and decanoic acid (B)
reactions (2 h) with MroUPO showing the remaining substrate (underlined),
the shortened monocarboxylic acids, and the α-hydroxy, (ω-1)-keto and
dicarboxylic derivatives.
Despite the difficulties for GC-MS estimation of initial rates in
the above MroUPO reactions, apparent kinetic constants could
be obtained for the products: i) resulting in chain-shortening; and
ii) of other oxygenation reactions (Table 1). Concerning
shortening, the MroUPO had higher catalytic efficiency (kcat/Km)
on C10 than on di-C14, due to the almost 10-fold higher catalytic
constant (kcat), although it was less selective as shown by the
ratios (0.8 and 2.0, respectively) between the catalytic
efficiencies of shortening and other reactions. The chain-
shortening of both mono- and dicarboxylic acids, and the α-
18
22
24
Retention time (min)
Figure 2. GC-MS of 1-h (A) and 24-h (B) reactions of tetradecanedioic acid
(underlined) with MroUPO, and with AaeUPO after 24 h (C), showing the
shortened dicarboxylic acids, and the α-, β-, and γ-hydroxylated and α-enol
derivatives (italics) (see mass spectra in Figures S1 and S2).
hydroxylation of carboxylic acids by
reported here for the first time.
a peroxygenase are
When monocarboxylic fatty acids were studied as MroUPO
substrates, the shortening reaction seemed to be chain-length
dependent. With tetradecanoic acid, terminal and subterminal
oxygenations (forming the dicarboxylic and [-1]-keto derivatives,
respectively) were predominant (Figure 3A). However, with
Table 1. Apparent kinetic constants for tetradecanedioic acid (di-C14)
and decanoic acid (C10) reactions with MroUPO
kcat
Km
(µM)
kcat/Km
(min-1)
(min-1·mM-1)
decanoic acid,
a relevant amount of nonanoic acid was
Chain shorteninga
di-C14
generated (Figure 3B), although the reaction was less selective
than with dicarboxylic acids.
32 ± 9
293 ± 98
557 ± 295
703 ± 549
58 ± 4
C10
420 ± 350
To explore the role of α-, β- and γ-hydroxy-derivatives as
intermediates in the chain-shortening mechanism, we studied
the reactions of α- and β-hydroxytetradecanoic acids with
MroUPO. With the α-hydroxy-derivative, tridecanoic acid was
one of the main products (Figure S3A), but no chain shortening
was accomplished with the β-hydroxy-derivative (Figure S3B).
This confirms that both even and odd chain fatty acids are
produced by successive removal of one carbon atom via α-
Other oxygenationsb
di-C14
C10
7 ± 1
337 ± 92
239 ± 42
28 ± 5
649 ± 430
520 ± 370
aAfter oxygenation at α position. bAt β, γ, ω and ω-1 positions.
To get additional insight into the chain shortening
mechanism, 18O-labeling studies with H218O2 (90% isotopic
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purity) were performed using tetradecanedioic acid as target
substrate (Figures S1 and S2). Overall, our data led to the
chain-shortening mechanism depicted in Scheme 1a. The initial
product of MroUPO reaction will be the α-hydroxy acid, as
demonstrated by incorporation of H218O2 oxygen to form α-
hydroxytetradecanedioic acid, whose diagnostic fragment (m/z
373, Figure S1A, top) appeared fully (90%) 18O-labeled (m/z 375,
Figure S1A, bottom). Its oxidation will yield a gem-diol (ketone
hydrate) from a second Cα hydroxylation by MroUPO that will be
in equilibrium with the ketone via dehydration, and then will react
with excess H2O2 decarboxylating and forming a new carboxyl
group (chain shortening) as explained below.
channel (Figure 4A) than AaeUPO (Figure 4B), the channel of
which is flanked by several bulky phenylalanine residues (a
narrow access channel also exists in CPO). This wider heme
channel directly exposes the reactive Fe=O of H2O2-activated
MroUPO (compound I) to the entering substrate, enabling
oxygenation at the α-position of carboxylic acids.
A
B
F191
Ac
Direct evidence for involvement of H218O2-borne oxygen into
the gem-diol/ketone formation yielding α-ketotetradecanedioic
could not be obtained, since α-keto acids rapidly decarboxylate
in the presence of oxidizing agents including H2O2.[15] However,
evidence of their transient formation was obtained, as the enolic
form was detected (Figures 2A, S3A and S5A). 18O-labeling also
illuminated the formation of tridecanedioic acid after
incorporation of two or one 18O-atoms (Figure S1B, bottom). The
co-existence of single and double 18O-labeling in the carboxylic
group suggests that a gem-diol, with some hydroxyl exchange
with the water (labelling loss), may occur prior to
decarboxylation and chain shortening. This second α-
hydroxylation is clearly provoked by the enzyme and not by the
H2O2, as revealed by the negative control with α-
hydroxytetradecanoic acid and H2O2 in the absence of enzyme
(Figure S6). However, the final reaction step can directly be
produced by the H2O2 present in the UPO reaction set-up,
mediated by a hydroperoxide intermediate, as reported for other
α-ketoacids.[16]
Ac
F160
F274
F69
F199
Figure 4. Different sizes of the heme-access channel in MroUPO (A) and
AaeUPO (B), and bulky phenylalanine residues (magenta) (an acetate
occupies the substrate-binding site). From 5FUJ (A) and 2YP1 (B).
Interestingly, one of the MroUPO crystal structures available
(5FUK) includes a bound palmitic acid molecule along the heme
access channel with one of the carboxylate oxygens at
coordination distance of the heme iron (Figure 5A) (while an
acetate occupies this position in 5FUJ, Figure 5B). Sub-terminal
oxygenation by most UPOs implies fatty-acid binding with the
carboxyl located at the channel entrance. However, the palmitic
acid position found in the 5FUK crystal is in agreement with the
unique chain-shortening ability reported here for MroUPO.
In summary, we show the ability of MroUPO to catalyze
The different reactivity of MroUPO and AaeUPO regarding α-
hydroxylation could be explained by the only recently available
crystal structure of MroUPO (PDB entries 5FUJ and 5FUK)
compared with the previously reported AaeUPO structure.[14]
MroUPO is a smaller protein but it has a wider heme-access
H2O2 H2O
H2O2 H2O
H2O2 H2O
H2O
O
O
O
O
O
O
1
1
a
b
c
d
R
R
R
R
R
R
R
R
O
R
+ CO2
( )
( )
( )
( )
( )
( )
HO
( )
( )
OH
OH
OH
OH
n
n
n
n
n
n
n
OH
O
O
H2O
OH
OH
O
O2 H2O2
H2O
NAD NADH
CoASH CoASCOCH3
O
OH
O
2
3
4
5
R
( )
( )
( )
n
n
n
n
SCoA
SCoA
SCoA
SCoA
SCoA
CoASCOCH3
NAD NADH
O
O
O
OH
R
R
6
7
8
R
R
R
R
R
( )
n
( )
( )
( )
n
n
n
n
SCoA
SCoA
O
OH
O
H2O2
O
O
9
9
R
+ CO2
+
( )
( )
( )
·
n
n
OH
OH
OH
Scheme 1. Comparison of fatty-acid shortening reactions: a) α-oxidation and decarboxylation by MroUPO; b) usual β-oxidation (multienzymatic) pathway; c)
α-oxidation (multienzymatic) pathway for β-methylated acids; and d) decarboxylation and alkene formation by P450; (R, H or COOH; enzymes: 1, MroUPO 2,
acyl-CoA oxidase; 3, enoyl-CoA hydratase; 4, 3-hydroxyacyl-CoA dehydrogenase; 5, 3-ketoacyl-CoA thiolase; 6, phytanoyl-CoA hydroxylase; 7, 2-hydroxy-
phytanoyl-CoA lyase; 8, aldehyde dehydrogenase; and 9, P450 fatty-acid decarboxylase).
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10.1002/chem.201704773
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the stepwise chain shortening of carboxylic acids through a
chemo-enzymatic reaction cascade (Scheme 1a). In plants,
fungi and animals, the general β-oxidation pathway, leads to
two-C shorter acids (Scheme 1b)[17] and the alternative α-
oxidation pathway (Scheme 1c), leading to one-C shorter fatty
acids, typically includes several steps (hydroxylation, activation,
cleavage of the C1-C2 bond and aldehyde dehydrogenation)
with several enzymes involved.[18] However, MroUPO is capable
of catalyzing all these reactions self-sufficiently (i.e. alone), in
the presence of H2O2. Bacterial P450s are also known to
decarboxylate fatty acids, but in this case n-1 terminal alkenes
(Scheme 1d), instead of chain-shortened fatty acids, are
formed.[19]
hydroxylation reactions,[12] using the protein engineering tools
recently applied to AaeUPO.[25]
Acknowledgements
This work was supported by the EnzOx2 (H2020-BBI-PPP-2015-
2-1-720297) EU-project, and the BIORENZYMERY (AGL2014-
53730-R) and NOESIS (BIO2014-56388-R) projects of the
Spanish MINECO (co-financed by FEDER). E.D. Babot (IRNAS)
is thanked for experimental help.
Keywords: alpha-oxidation • carboxylic acids • chain shortening
• oxidoreductases • peroxygenase • hydrogen peroxide
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A
Palmitic
acid
[4] A. T. Martínez, F. J. Ruiz-Dueñas, S. Camarero, A. Serrano, D.
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12251.
B
F160
Acetate
Mg2+
[13] M. Sundaramoorthy, J. Terner, T. L. Poulos, Structure 1995, 3,
1367-1377.
[14] K. Piontek, E. Strittmatter, R. Ullrich, G. Gröbe, M. J. Pecyna, M.
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[17] H. Schulz, in Biochemistry of Lipids, Lipoproteins and
Membranes ,Vol. Chapter 5, (Eds.: D. E. Vance, J. Vance),
Elsevier, Amsterdam 2008, p. pp. 131-154.
Figure 5. Palmitic acid (A) and acetate (B) ligands in MroUPO crystal
structures. Ligands, heme cofactor and neighbor Phe160 are shown as sticks
(CPK colors) while Mg2+ cations are shown as spheres (when palmitic acid is
present in the crystal, the Phe160 side-chain adopts two different
conformations). From PDB entries 5FUK (A) and 5FUJ (B).
[18] G. A. Jansen, R. J. A. Wanders, Biochimica et Biophysica Acta
(BBA) - Molecular Cell Research 2006, 1763, 1403-1412.
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This carbon-by-carbon chain-shortening reaction represents
novel chemistry that may be used in biotechnological
a
applications including the obtainment of tailor-made acids such
as odd-numbered dicarboxylic or monocarboxylic fatty acids
(less abundant in nature than the even-numbered ones). The
“odd-even” effect on the aqueous solubility of dicarboxylic
acids[20] could be used for product isolation, and in the synthesis
of ad-hoc polymers.[21]
The chain-shortening reaction described here must be
added to the repertoire of reactions that versatile fungal
peroxygenases catalyze on linear[12;22] and cyclic aliphatic
compounds,[23;24] in addition to aromatic compounds.[1;2] The
availability of a heterologous expression system for MroUPO will
permit to improve the catalytic properties of this promising
enzyme, e.g. in chain-shortening and/or alkane terminal
[24] F. Lucas, E. D. Babot, J. C. del Río, L. Kalum, R. Ullrich, M.
Hofrichter, V. Guallar, A. T. Martínez, A. Gutiérrez, Catal. Sci.
Technol. 2016, 6, 288-295.
[25] P. Molina-Espeja, M. Canellas, F. J. Plou, M. Hofrichter, F. Lucas,
V. Guallar, M. Alcalde, ChemBioChem 2016, 17, 341-349.
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Entry for the Table of Contents (Please choose one layout)
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A peroxygenase from the fungus
Marasmius rotula catalyzes the
stepwise chain shortening of
carboxylic acids by one carbon atom,
in the presence of H2O2 as the sole
Andrés Olmedo, José C. del Río, Jan
Kiebist, René Ullrich, Martin Hofrichter,
Katrin Scheibner, Angel T. Martínez,
Ana Gutiérrez*
MroUPO
α
Page No. – Page No.
co-substrate
O
H2O2
OH
R
R
Fatty acid Chain Shortening by a
Fungal Peroxygenase
()
()
OH
n
n
O
CO2 + H2O
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Author(s), Corresponding Author(s)*
((Insert TOC Graphic here))
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Title
Text for Table of Contents
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