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other fungi such as Fomes fomentarius [10], Pycnoporus
cinnabarinus [11], Aspergillus sp. [11, 12], Bjerkandera sp.
[13], Mucor sp. [14], and many others [15, 16]. However,
to date, the amino acid sequence of only one single fungal
carboxylate reductase has been elucidated from Aspergillus
terreus [17, 18]. Herein, the sequence and substrate scope
of the first carboxylate reductase from T. versicolor have
been explored.
the expression conditions, such as the choice of a weaker
promoter, the use of chaperones [23], or ultimately a fungal
expression system, will likely result in significant
improvements with respect to enzyme yield.
In comparison to published CAR enzymes, T. versicolor
CAR is most similar to Aspergillus terreus CAR, albeit
with only 24 % identity according to UniProtAlign,
whereas N. iowensis CAR and M. marinum CAR exhibit
17 % and S. rotundus CAR 16 % identity, respectively.
Carboxylate reductase enzymes rely on ATP and
NADPH as cosubstrates. As typical for adenylating
enzymes, the presence of Mg2? is important due to its
interaction with ATP [24]. TvCAR preparation was assayed
using the conditions as described in the experimental sec-
tion. In addition, control reactions without the addition of
substrate (E)-cinnamic acid (1a), ATP, or MgCl2 were
carried out. NADPH was not oxidized in the absence of
ATP or substrate. When the reaction was carried out in
TrisHCl buffer without MgCl2, the carboxylate reduction
rate was diminished by approximately 30 %. It needs to be
noted that the enzyme preparation contained 10 mM
MgCl2, resulting in 0.5 mM MgCl2 end concentration in
this particular reaction.
Results and discussion
Trametes versicolor (alias: Polystictus versicolor) was
reported to reduce benzoic acid derivatives to mixtures of
aldehydes and alcohols [9]. The enzyme responsible for the
first reduction step from the acid to the aldehyde had not
been identified to this date, nor has a particular enzyme
been purified and characterized. To discover the enzymatic
activity on sequence level, the fungal carboxylate reductase
enzyme sequence and three sequences of homologous
bacterial ATP and NADPH-dependent carboxylate reduc-
tases were used as templates to search within the non-
redundant protein sequences on NCBI with the restriction
to the organism T. versicolor. The hits from this search
were all annotated as acetyl-CoA synthetase-like proteins.
Aspergillus terreus ATEG_03630 [17] is classified as CaiC
for the adenylation domain and Thioester-redct for the
reduction domain by the multi-domain model, whereas the
CARs from Nocardia iowensis [19], Mycobacterium mar-
inum [20], and Segniliparus rotundus [21] are classified as
FAA1 and Lys2B. From the most significant hits of the
search, one sequence was classified to consist of an FAA1
adenylation domain in combination with a Thioester-re-
ductase domain. Therefore, it seemed most likely that this
sequence would display carboxylate reductase activity.
Since homologous bacterial carboxylate reductases were
shown to require the attachment of a phosphopantethein
moiety [22], the protein was expressed from the pET-
DUET1 vector that harbored simultaneously Escherichia
coli phosphopantethein transferase for post-translational
modification of the carboxylate reductase and the coding
sequence of the putative CAR enzyme with an N-terminal
fusion tag. Despite codon optimization of the fungal gene
sequence for expression in E. coli, the amount of soluble
protein was disappointingly low and the majority of the
protein was found in the insoluble fraction. The protein was
purified via affinity chromatography, yielding T. versicolor
CAR (TvCAR) in enriched form. As known CAR enzymes
have been well expressed heterologously in E. coli in their
apo-form [22], the reason is unlikely insufficient post-
translational phosphopantetheinylation. Optimization of
TvCAR was subjected to an NADPH depletion assay in
the presence of different carboxylic acids. Enzymatic
activity was observed for the substrates listed in Table 1. In
the presence of, e.g. phenylacetic acid, mandelic acid,
2-pyrrole-, 2-pyridine-, 3-indole-, and 2-pyrazinecar-
boxylic acid as well as short chain aliphatic acids levulinic
acid, pentanoic acid and hexanoic acid, NADPH was not
oxidized by TvCAR. The fact that 1a, benzoic acid (2a),
3-hydroxy- and 3-methoxybenzoic acid (3a, 4a) as well as
4-methoxybenzoic acid (5a) are reduced, but phenylacetic
acid is not, is consistent with the observations of Farmer
et al. [9]. However, 2-hydroxy- and 2-methoxybenzoic
acids were not reduced by TvCAR under the conditions
used. This supports the idea that T. versicolor harbors more
than one CAR enzyme and CAR (XP_008043822.1) is
partly but not fully responsible for the reactions observed
in 1959. As an analog of 1a, 3-phenylpropanoic acid (6a)
was also reduced. Surprisingly, also aliphatic carboxylates
from C7 to C9 (7a–9a) were converted, whereas short
chain acids and longer chain acids were not accepted as
substrates under these conditions (Table 1).
To investigate the identity of the reduction product, (E)-
cinnamic acid (1a) was subjected to a biotransformation
reaction in the presence of the TvCAR enzyme and excess
of co-factors. The reaction was analyzed after extraction
and derivatization of the remaining substrate 1a by GC/MS
analysis, revealing cinnamaldehyde (1b) as the sole pro-
duct in a mixture with methyl cinnamate (1d) (Scheme 1).
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Winkler, Margit
