The oxidation of thiols by flavoprotein oxidases: A biocatalytic route to reactive thiocarbonyls

Article abstract of DOI:10.1002/anie.201407520

Flavoprotein oxidases are a diverse class of biocatalysts most of which catalyze the oxidation of C-O C-N or C-C bonds. Flavoprotein oxidases that are known to catalyze the oxidation of C-S bonds are rare being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein we report that various flavoprotein oxidases previously thought to solely act on alcohols also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds which have a variety of applications in synthetic organic chemistry.

Full text of DOI:10.1002/anie.201407520

Angewandte
Chemie
DOI: 10.1002/anie.201407520
Biocatalysis
The Oxidation of Thiols by Flavoprotein Oxidases: a Biocatalytic
Route to Reactive Thiocarbonyls**
Tom A. Ewing, Willem P. Dijkman, Jacques M. Vervoort, Marco W. Fraaije, and
Willem J. H. van Berkel*
Abstract: Flavoprotein oxidases are a diverse class of biocat-
into aldehydes and thiols, whereas sulfhydryl oxidases
(EC 1.8.3.2 and 1.8.3.3) catalyze the formation of disulfide
bonds from thiols. So far, no flavoprotein oxidases have been
identified that catalyze the oxidation of thiols to thiocarbonyl
compounds. Herein, we present the finding that a number of
flavin-dependent oxidases, that hitherto had been thought to
act solely on alcohols, also oxidize thiols that are structurally
similar to their alcohol substrates, yielding the corresponding
thiocarbonyls.
À
À
alysts, most of which catalyze the oxidation of C O, C N, or
À
C C bonds. Flavoprotein oxidases that are known to catalyze
À
the oxidation of C S bonds are rare, being limited to enzymes
that catalyze the oxidative cleavage of thioethers. Herein, we
report that various flavoprotein oxidases, previously thought to
solely act on alcohols, also catalyze the oxidation of thiols
to thiocarbonyls. These results highlight the versatility of
enzymatic catalysis and provide a potential biocatalytic route
to reactive thiocarbonyl compounds, which have a variety of
applications in synthetic organic chemistry.
Alditol oxidase from Streptomyces coelicolor (AldO;
EC 1.1.3.41) is a flavin-dependent oxidase that catalyzes the
regioselective oxidation of the primary hydroxy groups of
a broad range of polyols, with the best substrates being
alditols, such as xylitol, d-sorbitol, and l-threitol.[4,5] AldO
displays stereoselectivity: for example, d-sorbitol is converted
into its oxidized form significantly more efficiently than its
C2 epimer d-mannitol. AldO belongs to the vanillyl alcohol
oxidase (VAO) family of structurally homologous flavopro-
teins. These flavoproteins contain a conserved FAD-binding
domain as well as a variable substrate-binding domain, which
determines their substrate specificity.[6] The enzyme contains
an FAD cofactor which is covalently linked to a histidine
residue.[7] To investigate whether AldO reacts with thiols, the
enzyme was first expressed and purified as a fusion protein
with an N-terminal maltose-binding protein tag. This fusion
protein (m-AldO) displayed identical catalytic properties to
the wild-type enzyme.[4] Oxygen consumption measurements
showed that m-AldO catalyzes the oxidation of dl-dithio-
threitol (dl-DTT), which is structurally identical to the known
AldO substrate threitol ((2R,3R/2S,3S)-butane-1,2,3,4-tetrol),
except for the presence of thiol rather than hydroxy groups at
the terminal carbon atoms. Steady-state kinetic analysis
revealed that m-AldO oxidizes racemic DTT with a turnover
number kcat = 0.79 sÀ1 and a Michaelis constant KM = 3.7 mm
(Table 1 and Figure S1 in the Supporting Information). The
l-enantiomer of DTT was preferentially converted into its
oxidized form, displaying a similar kcat value (0.71 sÀ1), but
a lower KM (2.4 mm) than the racemic mixture (Table 1 and
Figure S2). m-AldO also oxidizes dithioerythritol, but the
reaction with this epimer of DTT is less efficient, primarily
because of a higher KM value, and displayed substrate
inhibition with an inhibition constant Ki = 353 Æ 41 mm
(Table 1 and Figure S3). The tested thiols are remarkably
good substrates for m-AldO. The catalytic efficiency (kcat/KM)
for the oxidation of l-DTT is very similar to that for the
oxidation of its alcohol analogue l-threitol. In fact, of all
known m-AldO substrates, only xylitol and d-sorbitol display
higher catalytic efficiencies than l-DTT.[4,5]
F
lavoprotein oxidases constitute a diverse class of oxido-
reductases, which contain a flavin cofactor in the form of
flavin adenine dinucleotide (FAD) or flavin mononucleotide
(FMN). They catalyze the oxidation of a wide range of
substrates, with molecular oxygen functioning as the final
oxidant. Their catalytic cycle consists of two half reactions.
During the reductive half reaction two electrons are trans-
ferred from the substrate to the flavin cofactor, a process
which is generally believed to occur through the direct
transfer of a hydride anion to the N5 atom of the flavin.[1,2] In
the oxidative half reaction the flavin cofactor is reoxidized by
molecular oxygen, which is reduced to hydrogen peroxide.
Unlike many other oxidative enzymes, for example dehydro-
genases, oxidases do not require stoichiometric amounts of
expensive organic cofactors, making them interesting candi-
dates for biocatalytic exploitation. Most of the characterized
À
À
flavoprotein oxidases catalyze the oxidation of C O, C N, or
[3]
À
C C bonds to the corresponding double bonds. A small
number of flavoprotein oxidases are known to oxidize sulfur-
containing compounds. Prenylcysteine lyase (Enzyme Com-
mission (EC) number 1.8.3.5) and farnesylcysteine lyase
(EC 1.8.3.6) catalyze the oxidative cleavage of thioethers
[*] T. A. Ewing,[+] Prof. Dr. J. M. Vervoort, Prof. Dr. W. J. H. van Berkel
Laboratory of Biochemistry, Wageningen University
Dreijenlaan 3 (The Netherlands)
E-mail: willem.vanberkel@wur.nl
Homepage: http://www.bic.wur.nl
W. P. Dijkman,[+] M. W. Fraaije
Molecular Enzymology Group
Groningen Biomolecular Sciences and Biotechnology Institute
University of Groningen, Nijenborgh 4 (The Netherlands)
[+] These authors contributed equally to this work.
[**] This work was carried out within the BE-Basic R&D Program, for
which an FES subsidy was granted from the Dutch Ministry of
Economic Affairs, Agriculture, and Innovation (EL&I).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201407520.
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Table 1: Steady-state kinetic parameters for the oxidation of thiols and their alcohol
analogues by flavoprotein oxidases.
in absorbance was observed at l = 530 nm (Fig-
ure 1A, see Supporting Information for details). This
increase in absorbance at longer wavelengths is
indicative of the formation of a charge-transfer
complex between the thiolate anion form of l-DTT
and the FAD cofactor. Such flavin–thiol charge-
transfer complexes have been observed in various
members of the sulfhydryl oxidase family, though in
these cases it is due to an interaction with an active
site cysteine residue.[8,9] Subsequently, the absorb-
ance at l = 530 nm decays concomitantly with the
absorbance at l = 450 nm, suggesting the charge-
transfer interaction is broken by transfer of electrons
from l-DTT to the flavin cofactor (Figure 1B).
Enzyme-monitored turnover experiments under
aerobic conditions revealed that during steady-state
Enzyme[a] Substrate
kcat(app) [sÀ1
] KM(app) [mm] kcat(app)/KM(app)
[sÀ1 mmÀ1
]
m-AldO dl-dithiothreitol
m-AldO l-dithiothreitol
m-AldO l-threitol
m-AldO dithioerythritol
m-AldO erythritol
0.79Æ0.01 3.7Æ0.1
0.71Æ0.01 2.4Æ0.1
0.21
0.30
0.25
0.016
N.D.
0.60
9.6
6.3[b]
25[b]
0.45Æ0.02 29Æ3
N.D.
>100
HMFO
HMFO
HMFO
HMFO
GO
phenylmethanethiol
benzyl alcohol
2.1Æ0.1
3.5Æ0.8
13.5Æ0.3[b] 1.4Æ0.1[b]
(4-nitrophenyl) methanethiol 3.0Æ0.09 15Æ1.4
0.20
(4-nitrophenyl) methanol
1-thio-b-d-glucose
d-glucose
4.5Æ0.1
50Æ12
223Æ2
0.078Æ0.01 58
71Æ21
35Æ1
0.70
6.4
GO
Steady state kinetic parameters were determined at 258C in the following buffers:
m-AldO: KPi buffer (20 mm, pH 7.2); HMFO: KPi (50 mm, pH 8.0); GO: citrate
buffer (50 mm, pH 5.5). [a] m-AldO=maltose-binding-protein alditol oxidase fusion catalysis m-AldO is predominantly present as the
protein, HMFO=HMF oxidase, GO=glucose oxidase. [b] The kinetic parameters
charge-transfer complex (Figure 1C).
have been described elsewhere.[4,14] N.D.=not determined.
HPLC analysis revealed that three main products are
formed during the oxidation of dl-DTT by m-AldO (Fig-
ure S4). Addition of the hydrogen peroxide scavenger sodium
pyruvate to the reaction mixture demonstrated that two of the
products are formed by side reactions involving hydrogen
peroxide. The third compound, which is the product of the
enzymatic reaction, was collected and identified by NMR
spectroscopy as the dithiohemiacetal 2 (Scheme 1, showing
Figure 1. The change in UV/Vis absorption intensity with time mea-
sured using a stopped-flow apparatus after mixing m-AldO (10 mm)
with l-DTT (25 mm) under anaerobic (A, B) or aerobic (C) conditions.
The absorption intensity was monitored at l=530 nm (solid lines)
and l=450 nm (dashed line). Time periods for each plot: A) t=
0–25 ms; B) t=0–15 s; C) t=0–50 s. Measurements were performed
at least three times, representative traces are shown.
Scheme 1. The oxidation of l-DTT catalyzed by alditol oxidase (AldO).
The VAO family, to which AldO belongs, displays
a remarkably broad range of reactivities. The oxidation of
À
the l-enantiomers, and Figure S5). Formation of this product
presumably occurs via the thioaldehyde intermediate 1, which
is formed upon oxidation of dl-DTT by m-AldO and
subsequently undergoes an intramolecular cyclization to
yield 2. This dithiohemiacetal is relatively unstable, being
entirely oxidized to the corresponding sulfenic acid after
6 days of storage at À208C dissolved in acetonitrile. By
comparison to a reference compound, one of the two side
products was identified as the disulfide trans-4,5-dihydroxy-
1,2-dithiane, which was shown to be formed upon reaction of
dl-DTT with hydrogen peroxide. The second unidentified
compound is likely the disulfide formed after reaction of two
molecules of 2 upon its oxidation by hydrogen peroxide.
Alternatively, it may be the mixed disulfide formed between
one molecule of 2 and one molecule of DTT.
alcohols and amines, hydroxylation of alkyl groups, C C bond
formation, and the demethylation of ethers have all been
described previously.[10–13] The results presented above show
that the oxidation of thiols can be added to this list. To
determine whether the oxidation of thiols is also performed
by more strict alcohol oxidases from the glucose-methanol-
choline (GMC) family, we investigated the recently discov-
ered 5-hydroxymethylfurfural oxidase (HMFO) from Meth-
ylovorus sp. Strain MP688.[14] HMFO catalyzes the oxidation
of primary alcohols. Its substrate range includes phenylic,
furanic, and linear unsaturated alcohols. In addition, HMFO
is active towards several aldehydes. However, this activity is
restricted to aldehydes which are spontaneously hydrated to
the gem-diol form when dissolved in water.[15]
We tested HMFO for activity towards phenylmethane-
thiol, which is the thiol analogue of its known substrate benzyl
alcohol, and the related (4-nitrophenyl)methanethiol. Inter-
estingly, both thiols were readily oxidized by HMFO. The
steady-state kinetic parameters of HMFO for these two
aromatic thiol substrates are shown in Table 1 and Figures S6
To further investigate the reaction of m-AldO with
l-DTT, changes in the spectral properties of the enzymeꢀs
flavin cofactor during the reductive half reaction were
followed using a stopped-flow apparatus. Upon mixing m-
AldO and l-DTTunder anaerobic conditions, a rapid increase
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and S8. The kcat values for both aromatic thiols are slightly
lower than those for their alcohol counterparts (Table 1,
Figures S7 and S9). Also, the KM values for both thiols are
significantly higher. Taken together, the catalytic efficiency of
HMFO is higher for alcohols than for thiols. Still, both tested
aromatic thiols can be regarded as good substrates with
kcat values greater than 2 sÀ1 and KM values in the millimolar
range.
The products formed upon oxidation of the two aromatic
thiol substrates by HMFO were identified using GC–MS and
LC–MS. At pH 7.0, phenylmethanethiol is oxidized to the
corresponding aromatic thioaldehyde, benzothialdehyde (3)
(Scheme 2 and Figures S10 and S11). No formation of 1,2-
dibenzyldisulfane was observed, demonstrating that HMFO
(Table 1 and Figure S12). However, the oxidation of 1-thio-
b-d-glucose by GO is less efficient than that of its alcohol
analogue d-glucose, which occurs with a higher kcat value
(223 sÀ1) and a lower KM value (35 mm) (Table 1 and Fig-
ure S13). Methanol oxidase (MO, or alcohol oxidase;
EC 1.1.3.13) is another fungal GMC-type oxidase. MO
contains a noncovalently bound FAD cofactor and catalyzes
the oxidation of linear unbranched primary alcohols of up to
5 carbon atoms in length.[19] MO from Candida boidinii was
shown to oxidize ethanethiol, displaying a specific activity of
0.32 Æ 0.03 UmgÀ1 at a substrate concentration of 20 mm, as
compared to a specific activity of 0.51 Æ 0.02 UmgÀ1 for the
oxidation of 20 mm ethanol.
In summary, all tested flavin-dependent alcohol oxidases
are capable of catalyzing the oxidation of thiols, yielding
thiocarbonyls. Though various free flavins are also known to
react with some thiols, these reactions solely yield disulfides
as products, suggesting that they are mechanistically distinct
from the flavoenzyme-catalyzed reactions described
herein.[20,21] In the reaction of m-AldO with l-DTT, the
thiol substrate binds to the enzyme as a thiolate anion, leading
to a strong charge-transfer interaction with the oxidized flavin
cofactor. Binding of the substrate in its thiolate form is likely
stimulated by the presence of two positively charged residues:
R322 and K375. These residues are in close proximity to the
terminal hydroxy group of the substrate in the crystal
structure of AldO with xylitol bound.[7] Subsequently, the
flavin is slowly reduced by the transfer of two electrons from
the substrate, yielding the thiocarbonyl product. The electron
transfer probably occurs through the direct transfer of
a hydride anion from the Ca atom of the substrate to the
N5 atom of the flavin cofactor, as is believed to be the case for
the oxidation of alcohols and amines by flavoprotein oxi-
dases.[1,2] In the case of the oxidation of DTT by m-AldO, the
formed thioaldehyde rearranges intramolecularly to yield
dithiohemiacetal 2. For the aromatic thiols oxidized by
HMFO, such a rearrangement is not possible because of the
absence of a second thiol group and in the case of phenyl-
methanethiol the formed thioaldehyde 3 is stable enough to
be detected. Because HMFO has an exceptionally broad
substrate range, this biocatalyst can be used to generate
a large variety of reactive, but relatively stable, thioaldehydes.
The fusion protein m-AldO also has a broad substrate range
and could potentially be used to generate more stable
thioaldehydes in cases where no second thiol group is present
to enable intramolecular cyclization. As the ability to oxidize
thiols appears to be a general property of flavin-dependent
oxidases, many other enzymes may be used for similar
purposes. Thus, our results not only highlight the promiscuity
and versatility of enzymatic catalysis, but also provide
a potential biocatalytic route to reactive thiocarbonyl com-
pounds, which have a variety of applications in synthetic
organic chemistry.[22]
Scheme 2. HMFO-catalyzed oxidation of phenylmethanethiol to benzo-
thialdehyde (3) and benzaldehyde (4).
does not catalyze the formation of disulfide bonds. At pH 8.0,
two products are formed. Although after 10 minutes only
benzothialdehyde was formed, after 60 minutes benzaldehyde
(4) was also detected. This suggests that the thioaldehyde is
slowly hydrated, yielding the aldehyde as the final product.
This is analogous to the reaction performed by amine
oxidases, where the oxidation of amines to imines is followed
by their reaction with water, yielding aldehydes or ketones.[16]
To confirm the involvement of water, the reaction at pH 8.0
was performed in the presence of 18O-labeled water. In the
presence of H218O, benzaldehyde with m/z 108 was observed,
whereas in unlabeled water only m/z 106 was detected. This
confirms that at pH 8.0, water performs a nucleophilic attack
on the initially formed thioaldehyde, yielding benzaldehyde
as the final product. Using (4-nitrophenyl)methanethiol as
a substrate, the reaction at pH 8.0 again yielded the corre-
sponding aldehyde. Because this aldehyde is easily hydrated
to the gem-diol,[17] it is oxidized further by HMFO, yielding
4-nitrobenzoic acid as the final product. In addition to these
main products, several minor peaks were detected by GC and
LC analysis, probably resulting from the reactive nature of
the formed thioaldehyde.
To determine whether the ability to oxidize thiols as well
as alcohols is a general property of flavin-dependent alcohol
oxidases, we investigated a number of commercially available
oxidases for their reactivity towards thiols. Glucose oxidase
(GO; EC 1.1.3.4) is a fungal glycoprotein, which also belongs
to the GMC-family and is widely applied as an oxidative
biocatalyst. Comparable to HMFO, GO contains a noncova-
lently bound FAD cofactor. GO regioselectively oxidizes
b-d-glucose to 1,5-d-gluconolactone, which spontaneously
hydrolyzes to gluconic acid.[18] Oxygen consumption measure-
ments showed that GO from Aspergillus niger indeed oxidizes
1-thio-b-d-glucose with a kcat value of 50 sÀ1, KM = 71 mm, and
with substrate inhibition occurring with Ki = 42 Æ 13 mm
Received: July 23, 2014
Revised: September 9, 2014
Published online: && &&, &&&&
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Keywords: biocatalysis · flavoprotein oxidases · kinetics ·
oxidoreductases · thiol oxidation
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4
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Communications
Biocatalysis
T. A. Ewing, W. P. Dijkman, J. M. Vervoort,
M. W. Fraaije,
W. J. H. van Berkel*
&&&& — &&&&
À
An all-around player: Numerous classical
flavin-dependent alcohol oxidases, such
as alditol oxidase (AldO), which are well-
C N bonds, can also catalyze the oxida-
tion of thiols (see picture). This method
provides a potential biocatalytic route to
reactive thiocarbonyl compounds.
The Oxidation of Thiols by Flavoprotein
Oxidases: a Biocatalytic Route to Reactive
Thiocarbonyls
À
known for their activity towards C O and
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