Biocatalytic Methyl Ether Cleavage: Characterization of the Corrinoid-Dependent Methyl Transfer System from Desulfitobacterium hafniense

Article abstract of DOI:10.1002/adsc.201801590

The ether functionality represents a very common motif in organic chemistry and especially the methyl ether is commonly found in natural products. Its formation and cleavage can be achieved via countless chemical procedures. Nevertheless, since in particular the cleavage often involves harsh reaction conditions, milder alternatives are highly demanded. Very recently, we have reported on a biocatalytic shuttle catalysis concept for reversible cleavage and formation of phenolic O-methyl ethers employing a corrinoid-dependent methyl transferase system from the anaerobic organism Desulfitobacterium hafniense. Here we report the technical study of this system, focusing on the demethylation of guaiacol as model reaction. The optimal buffer-, pH-, temperature- and cofactor-preferences were determined as well as the influence of organic co-solvents. Beside methyl cobalamin also hydroxocobalamin turned out to be a suitable cofactor species, although the latter required activation. Various O-methyl phenyl ethers were successfully demethylated with conversions up to 82% at 10 mM substrate concentration. (Figure presented.).

Full text of DOI:10.1002/adsc.201801590

Title: Biocatalytic Methyl Ether Cleavage: Characterization of
the Corrinoid-Dependent Methyl Transfer System from
<i>Desulfitobacterium hafniense</i>
Authors: Nina Richter, Judith Farnberger, Simona Pompei, Christopher
Grimm, Wolfgang Skibar, Ferdinand Zepeck, and Wolfgang
Kroutil
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
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
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801590
Link to VoR: http://dx.doi.org/10.1002/adsc.201801590

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
UPDATE
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Biocatalytic Methyl Ether Cleavage: Characterization of the
Corrinoid-Dependent Methyl Transfer System from
Desulfitobacterium hafniense
Nina Richter,a,§ Judith E. Farnberger,a,§ Simona Pompei,b Christopher Grimm,b
Wolfgang Skibar,c Ferdinand Zepeck,c and Wolfgang Kroutilb*
a
Austrian Centre of Industrial Biotechnology, ACIB GmbH, c/o University of Graz, Heinrichstrasse 28, 8010 Graz,
Austria
b
Institute of Chemistry, University of Graz, NAWI Graz, BioTechMed Graz, Heinrichstrasse 28, 8010 Graz, Austria
[Fax: (+43)-316-380-9840; phone: (+43)-316-380-5350; e-mail: wolfgang.kroutil@uni-graz.at]
Sandoz GmbH, Biocatalysis Lab, Biochemiestrasse 10, 6250 Kundl, Austria
c
§ These authors contributed equally
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
in nature, where O-demethylation is part of many
Abstract. The ether functionality represents a very
common motif in organic chemistry and especially the aspects of life.[16-17] Particularly, oxidative
enzymes[18-21] such as fungal peroxygenases[22-23] and
methyl ether is commonly found in natural products. Its
bacterial monooxygenases[24-25] including P450s[26-27]
formation and cleavage can be achieved via countless
chemical procedures. Nevertheless, since in particular the
cleavage often involves harsh reaction conditions, milder
alternatives are highly demanded. Very recently, we have
reported on a biocatalytic shuttle catalysis concept for
reversible cleavage and formation of phenolic O-methyl
ethers employing a corrinoid-dependent methyl transferase
system from the anaerobic organism Desulfitobacterium
hafniense. Here we report the technical study of this
system, focusing on the demethylation of guaiacol as model
reaction. The optimal buffer-, pH-, temperature- and
cofactor-preferences were determined as well as the
influence of organic co-solvents. Beside methyl cobalamin
also hydroxocobalamin turned out to be a suitable cofactor
species, although the latter required activation. Various O-
methyl phenyl ethers were successfully demethylated with
conversions up to 82% at 10 mM substrate concentration.
are known to catalyze O-dealkylation reactions. They
are primarily involved in the biodegradation of
lignocellulosic compounds, detoxification of organic
chemicals and alkaloid biosynthesis. Apart from that,
specific O-demethylases have been described to
occur in various anaerobic organisms[28-30] utilizing
methyl aryl ethers as carbon and energy source.
Considering this vast number of biocatalysts capable
of performing ether cleavage, it is astonishing that
with exception of a few examples[31-33] hardly any of
them have been applied for synthetic purposes, yet.
This
is
in
contrast
to
SAM-dependent
methyltransferases which are broadly investigated,
however exclusively for alkylation.[34-36] An
alternative group of enzymes which may be
considered for ether cleavage are corrinoid-dependent
methyltransferases (MTases)[37-38] which have
recently been used for both O-methylation as well as
demethylation.[39] The reaction involves a two-
component methyl transfer system consisting of a
corrinoid protein (CP) carrying the cobalamin
cofactor[40-42] and a MTase from Desulfitobacterium
hafniense (D. hafniense).[43] This system allows
performing both demethylation of a donor molecule
and subsequent methylation of an acceptor in a
reversible fashion. The CP with the cobalt containing
cofactor acts as shuttle for the methyl group with
cobalt cycling between the nucleophilic CoI and the
CoIII state. By using an excess of the methyl acceptor
as co-substrate, demethylation of phenyl ethers can
be performed. Here, we present now the
characterization of the recently published MTase
system from D. hafniense,[39] reporting on the
influence of various parameters on the reaction and
Keywords: Biocatalysis, Biotransformations, Ether
Cleavage, Demethylation, Methyl transferases, Corrinoids
Introduction
Ether cleavage[1] is a valuable integral group
transformation in synthetic organic chemistry,
frequently used as a deprotection step to unmask
hydroxyl moieties.[2-3] Since O-methyl groups are
rather stable and thus more resistant to
hydrolysis/cleavage, classical procedures for the
cleavage of C-O-bonds of methyl ethers usually
involve the presence of strong Bronsted[4-7] or Lewis
acids.[8-10] Alternatively, the use of metal catalysts[11-
12]
or nucleophilic methods employing sodium
thiolates[13-15] belong to the most commonly applied
chemical techniques. Ether cleavage is also prevalent
1
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
its unique properties focusing here especially on
demethylation.
and methylation are reversible reactions but for the sake of
Results and Discussion
The transfer of a methyl group from the model
substrate guaiacol 1a (Scheme 1) onto a non-natural
methyl acceptor such as 3,4-dihydroxybenzoic acid
2b has been successfully proven in a reversible
shuttle catalysis concept[39] using a corrinoid-
dependent MTase system from the anaerobic
bacterium D. hafniense. Alternatively, aldehyde 2c
was used as methyl accepting co-substrate in 5-fold
molar excess in order to push the reaction equilibrium
towards the product side. Since the used corrinoid
cofactor is known to be highly oxygen-sensitive,
biotransformations were performed under inert
atmosphere. An inadvertently formed CoII was
reduced back to the active CoI species using a
chemical reactivation system composed of titaniumIII
citrate and methyl viologen (MV).
As a first step the catalytic system was characterized
with regard to preferred buffer and pH conditions,
enzyme amount as well as its optimal temperature
range (Figure 1 and Figure S1). While the enzymes
showed to have their pH optimum at neutral pH, high
conversions (up to 75%) were still achieved under
slight basic conditions (pH 8) when using MOPS-
KOH buffer.
clarity for this study the arrows just point in one direction.
Figure 1. Characterization studies addressing reaction
conditions. (A) Effect of varied buffers, salts and pH
values on the conversion of guaiacol. (B) Temperature
profile of the reaction giving relative initial rates (bars) and
conversions after 24 h (-♦-). Reaction conditions if not
indicated otherwise in respective studies: substrate 1a (10
mM, 1.2 mg/mL), methyl acceptor 2b (50 mM, 7.7
mg/mL), MTase I (40 mg/mL lyophilized cell-free crude
extract CFE), CP (400 µL/mL reconstituted holo-CP
solution), activation system (4.19 mM TiIII citrate and 0.3
mM methyl viologen) in MOPS buffer (50 mM, pH 6.5,
150 mM KCl) at 30 °C, 800 rpm in in Eppendorf Orbital
Shaker (1.5 mL) for 24 h. Conversions were determined
after 24 h by HPLC-UV from areas of 1a and 2a using
calibration curves. Reaction rates were determined from
the linear slopes within 0-2 hours of the reaction time
courses and calculated relatively to the highest one
Scheme 1. Model reaction for characterization of
biocatalytic corrinoid-dependent methyl transfer.
MTase I and CP from D. hafniense catalyze the
demethylation of guaiacol by simultaneously transferring
the methyl group onto the co-substrate (3,4-dihydroxy-
benzoic acid or related aldehyde) acting as methyl acceptor. obtained in each case.
While MTase I catalyzes both methyl transfers, CP
resembles a molecular shuttle for the methyl group,
switching between the super-reduced CoI and the CoIII state. With respect to the temperature optimum, increased
In order to reactivate an inadvertently formed CoII species
a chemical activation system based on titaniumIII citrate
and methyl viologen (MV) was used. Both demethylation
temperatures of up to 40 °C significantly enhanced
the initial reaction rate, however, enzyme inactivation
over time led to reduced overall conversion at 40 °C
and to a loss of activity at 45 °C. The methyl transfer
2
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
reaction is depending on the MTase catalyzing the
demethylation itself as well as on the CP carrying the
prosthetic group and shuttling the methyl group
between substrate and co-substrate. Since CP is
produced by E. coli as apo-protein devoid of its
cofactor, because E. coli does not produce cobalamin,
a reconstitution with Me-Cbl is required after
expression to obtain active holo-CP. Various
reconstitution protocols were examined (see Figure
S2), showing that reconstitution occurs rather fast (no
extended incubation time needed) probably because
the cobalamin-binding domain is located on the
surface of CP.[44-45] In addition to methyl cobalamin
(Me-Cbl), other cobalamin derivatives with varied β-
axial ligands at the central cobalt atom were
examined (Figure 2). Since all of them bear cobalt in
oxidation state three, the chemical reducing agent TiIII
is required together with MV for activation in order
to get cobalt in the demanded oxidation state (I).
While cyano- (CN-Cbl) and 5´-deoxyadenosyl
cobalamin (Ado-Cbl) led to a significantly reduced
demethylation activity, hydroxocobalamin (OH-Cbl)
was equally well accepted as Me-Cbl with a
comparable conversion of 80% and a relative initial
reaction rate of 93% (Figure 2). While Me-Cbl can
directly enter the catalytic cycle as methyl donor to
yield the reduced species during the first methyl
group transfer onto the acceptor molecule, a two-
electron-reduction of the CoIII has to precede in case
of OH-Cbl before it can act as nucleophile to pick up
the first methyl group. In-vitro activation of the CP-
bound cofactor is promoted by TiIII citrate in
combination with the redox mediator MV, which
actuates also as O2-scavenger system and reduces
from E. coli (cell free extract). Since the reaction
employing Me-Cbl worked well under inert
atmosphere even in the absence of TiIII/MV, we
investigated whether each step of the reaction had to
be handled under strictly anaerobic conditions. Thus,
the sensitivity of the reaction mixture towards
oxygen/air was examined as well as the use of
TiIII/MV for reactivation of reactions that had been
exposed to air. For this purpose, a series of identical
biotransformation reactions were prepared under inert
conditions and then treated differently with respect to
incubation, oxygen input and reactivation (Figure 4).
While more than 30% of substrate has been converted
after 2 hours under inert conditions (entry 1),
conversion tremendously decreased (2%) when the
reaction mixture was oxygenated right after its
preparation (entry 2), most likely due to oxidation of
the catalytic CoI species. Nevertheless, it can be
assumed that the enzymatic system was not
completely inactivated since some demethylation
product (16%) was still observed after 24 h despite of
occurred oxygen-input and incubation under aerobic
conditions. Interestingly, reactions which were
incubated on air but only ventilated after 2 h (entry 3-
6) gave comparable conversions (27-29%) as the
oxygen-free counterpart, indicating that almost no
oxygen entered the reaction tubes without actively
opening and mixing them. The effects of O2-input
turned out to be severe though, since it entirely
stopped the demethylation reaction, no matter if
incubation afterwards was performed on air (entry 3)
or in the glove-box (entry 4). However, a repeated
inadvertently
oxidized
cofactor.[46]
Suitable
concentrations of both compounds have been
determined for Me-Cbl-mediated reactions (Table S1)
and subsequently the requirements of TiIII/MV were
compared with the OH-Cbl-mediated demethylation
of guaiacol (Figure 3). The experiments employing
Me-Cbl clearly show that TiIII/MV is not required in
this case as long as the transformation can be
performed under strictly oxygen free conditions
(Figure 3, entry 1-2). For OH-Cbl mediated reactions,
almost no activity was observed in the absence of TiIII
(entry 5-6), thus TiIII turned out to be the crucial
reagent needed for CP activation. MV seemed to have
a supporting function since in presence of both
compounds (entry 3) the reaction rate almost reached
the same level as observed with Me-Cbl-CP (entry 1).
Interestingly, despite the significant differing initial
rates observed depending on the presence and
absence of TiIII and MV, comparable conversions in
the range of 74-84 % were obtained in all cases when
using a crude enzyme preparation. In contrast, when
performing the OH-Cbl mediated reaction with
purified enzyme (entry 8-9), activity was only
observed in the presence of the chemical activation
system reaching a comparable initial rate as obtained
with Me-Cbl (entry 7). Therefore, it can be assumed
that there are alternative pathways for OH-Cbl
activation in crude enzyme preparations originating
addition of O2-scavenger system (entry 5-6) showed
to actuate the reaction again and increased
conversions up to 41-42%. These results demonstrate
the option to reactivate oxidized cofactor by chemical
reduction, even though the highest efficacy of the
enzymatic system could not be regained.
Figure 2. Examination of the cofactor preference of CP.
Reconstitution: Incubation of apo-CP (100 mg/mL
lyophile. crude extract) in 1 mL TRIS-HCl buffer (50 mM,
pH 7, 0.5 mM DTT and 0.1 mM PMSF) with 2 mM of the
respective cobalamin species and 3 M betaine, followed by
a desalting step into MOPS buffer. Reaction conditions: 1a
(10 mM, 1.2 mg/mL), 2b (50 mM, 7.7 mg/mL), MTase I
and CP (40 mg/mL lyophilized crude extract of MTase I
and 400 µL/mL reconstituted holo-CP solution), activation
system (4.19 mM TiIII citrate and 0.3 mM methyl viologen)
in MOPS buffer (50 mM, pH 6.5, 150 mM KCl) at 30 °C,
3
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
800 rpm in in Eppendorf Orbital Shaker (1.5 mL) for 24 h.
Conversions were determined after 24 h by HPLC-UV
from areas of 1a and 2a using calibration curves. Error
bars represent the standard error of the mean (s.e.m., nꢀ=ꢀ3).
Reaction rates were determined from the linear slopes
within 0-2 hours of the reaction time courses and
calculated relatively to the highest one obtained in each
case.
Figure 3. Characterization study addressing the use of TiIII citrate and methyl viologen (MV) for in-vitro activation
of OH-Cbl to the superreduced CoI species. Reaction conditions: substrate 1a (10 mM, 1.2 mg/mL), methyl acceptor 2b
(50 mM, 7.7 mg/mL), MTase I (40 mg/mL freeze-dried CFE or 13 mg/mL freeze-dried pure enzyme ~0.36 mM,
respectively), CP (400 µL/mL holo-CP solution; 100 mg/mL freeze-dried CFE or 33 mg/mL pure apo-CP reconstituted
either with Me-Cbl or OH-Cbl, respectively), chemical activation system composed of TiIII citrate (as indicated, 1x refers
to 4.19 mM) and MV (as indicated, 1x refers to 0.3 mM) in MOPS buffer (50 mM, pH 6.5, 150 mM KCl) at 30 °C, 800
rpm in in Eppendorf Orbital Shaker (1.5 mL). Conversions were determined after 24 h by HPLC-UV from areas of 1a and
2a using calibration curves. Error bars represent the standard error of the mean (s.e.m., nꢀ=ꢀ3). Initial reaction rates were
determined from the linear slopes within 0-2 hours of the reaction time courses (for examples see Figure S4) and
calculated relatively to the highest one obtained in each case.
It is worth to mention that the proteins on their own
(without Cbl) can be stored at 4°C at least for 12
months since the activity of the methyltransferase
system is afterwards still unchanged.
conditions involving the presence of 10 % v/v co-
solvent (DMSO or MeOH, respectively) were applied
for the transformation of a selection of O-methyl
phenyl ethers (Table 1, entry 2-6). Vanillyl alcohol as
well as its regioisomer isovanillyl alcohol
(compounds m- and p-1d, entry 2-3, m = meta, p =
para refers to the position of the methyl group in
relation to the substituent at the catechol chore) were
demethylated with conversions up to 82%.
Comparable results were obtained for the bis-methyl
substrates 2,3-dimethoxyphenol 2,3-1e (up to 71%;
entry 4) and 2,6-1e (up to 81%, entry 5). In case of
the latter substrates a demethylation reaction can take
place at two sites; a single methyl transfer leads to the
formation of 3-methoxycatechol 2e which is further
demethylated to 1,2,3-trihodroxybenzene 3e.
Therefore, a product mixture was obtained in both
cases, with a slight surplus of 3e.
Finally, the effect of co-solvent addition was
investigated since for substrates that are barely
soluble in water, the addition of organic co-solvent
might be helpful to increase their solubility. First
experiments testing the model reaction in presence of
dimethyl sulfoxide (DMSO) showed that up to 20%
v/v of co-solvent are tolerated by the enzymatic
system from D. hafniense without significant loss of
activity (data not shown). Besides DMSO also
methanol (MeOH) was tolerated equally well with
conversions identical to the co-solvent free
counterpart (Table 1, entry 1). Furthermore, the
addition of dimethylformamide (DMF), ethanol
(EtOH) and 2-propanol (i-PrOH) has proven to be
possible leading to slightly decreased conversions in
the range of 33 to 56%. Finally, the optimized
Conclusion
4
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
A corrinoid-dependent methyl transfer system from
D. hafniense was shown to be applicable for the
demethylation of selected methyl phenyl ethers. The
reaction works in a broad operational window
concerning pH (pH 6.5-8) and temperature (25-
40 °C), whereby ideally the reaction is performed at
neutral pH and 30-35 °C. Reconstitution of CP with
the corrinoid cofactor has been shown to occur
spontaneously and to be crucial for an efficient
reaction. Methyl- and hydroxocobalamin (Me-Cbl,
OH-Cbl) turned out to be the suitable cofactor species.
In an inert atmosphere the addition of a chemical
activation system for reducing inadvertently oxidized
cobalamin (CoII) can be omitted. The reaction can be
performed in presence of various organic co-solvents
such as DMSO and MeOH without loss of activity at
10% v/v. The conditions reported in this study were
successfully employed for the demethylation of a
range of methyl phenyl ethers indicating that this
transformation may become a suitable reaction to be
considered for biocatalytic retrosynthesis.[47-50]
Figure 4. Reactivation of reactions exposed to air. A series of identical biotransformation reactions was prepared under
inert conditions. Reaction no.1 was entirely kept under an oxygen-free atmosphere as a control, whereas no.2-6 were
incubated for at least 2 h under aerobic conditions (closed vials on air). Samples were actively aerated either directly (0 h,
no. 3) or after 2 h incubation time (no. 3-6) by repeatedly opening the respective vials on air and mixing. While incubation
was continued under aerobic conditions in case of reaction no.3, reactions no. 4-6 were again transferred back into the
glove-box and incubated under inert atmosphere for the remaining 22 h. The O2-scavenger system was added to reaction
no.5 (1x) and no.6 (2x). Reaction conditions: substrate 1a (10 mM, 1.2 mg/mL), methyl acceptor 2b (50 mM, 7.7 mg/mL),
MTase I and CP (40 mg/mL lyophilized crude extract of MTase I and 400 µL/mL reconstituted holo-CP solution), O2-
scavenger system (1x relates to 4.19 mM TiIII citrate and 0.3 mM methyl viologen) in MOPS buffer (50 mM, pH 6.5, 150
mM KCl) at 30 °C, 800 rpm in in Eppendorf Orbital Shaker (1.5 mL) for 24 h. Conversions were determined after 2 h and
24 h by HPLC-UV from areas of 1a and 2a using calibration curves. Error bars represent the standard error of the mean
(s.e.m., nꢀ=ꢀ3).
5
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
performed as described before.[39] If necessary, enzymes
were purified by affinity chromatography, using Strep-
Tactin® technology (IBA Lifesciences) according to the
manual provided by the supplier (see SDS gel of
overexpressed and purified enzymes in Figure S3).
Biocatalysts were used as freeze-dried cell free extracts
(CFE) or purified preparations and biotransformations
were performed at least in triplicates in 1.5 mL Eppendorf
tubes using an Eppendorf Thermomixer comfort. Due to
the oxygen-sensitive cofactor, biocatalytic reactions were
performed in degassed buffers under inert atmosphere (N2
5.0) in a MBraun LABstar glove box equipped with a MB-
OX-EC O2-sensor. For detailed analytical methods and
NMR-spectra see electronic Supplementary Information.
Table 1. Effect of organic co-solvents on the corrinoid-
dependent demethylation of selected substrates.
Representative procedure for preparation of holo-CP.
The corrinoid protein was reconstituted with exogenous
cofactor under inert atmosphere in order to obtain
functional holo-CP. For this purpose, the respective
cobalamin species (2 mM) was dissolved in the presence of
betaine (3 M) and DTT (2mM) in TRIS/HCl buffer (50
mM, pH 7, 0.5 mM DTT, 0.1 mM PMSF). Then, freeze-
dried CP (100 mg/mL CFE or 33 mg/mL purified enzyme,
respectively) was dissolved in the prepared reconstitution
buffer (1 mL) and incubated for 2 h at 4 °C to allow
incorporation of the cofactor. Afterwards salts and
unbound cobalamin were removed using a PD MidiTrapTM
G-25 column (GE Healthcare) or PD 10TM G-25 column
(GE Healthcare) according to the manual provided by the
manufacturer. Finally, reconstituted holo-CP was eluted
with MOPS/KOH buffer (100 mM, pH 6.5, 150 mM KCl)
yielding a red colored protein fraction (containing 66.7
mg/mL CFE or 22 mg/mL pure enzyme, respectively)
which was stored at 4 °C until further use. The procedure
was adapted according to the performed investigations (e.g.
incubation times, cofactor species).
Representative procedure for biocatalytic demethyl-
ation. Analytical biotransformation reactions were carried
out on 180 µL or 1 mL scale as follows: freeze-dried
MTase I (10-20 mU or 56-112 mU, 40 mg/mL CFE or 13.3
mg/mL pure enzyme, respectively; ratio MTase I to
substrate: 1:30) was rehydrated in holo-CP solution (400
µL/mL). The reaction was started by adding appropriate
amounts of 1a (final concentration 10 mM) and methyl
acceptor 2b/2c (final concentration 50 mM) as stock
solutions in MOPS/KOH buffer (50 mM, pH 6.5, 150 mM
KCl). In case the addition of an activation system was
required, TiIII chloride (final concentration 4.15 mM) was
added as a stock solution in MOPS/KOH (1M, pH 7.9, 333
mM sodium citrate) to the reaction mixture as well as MV
(0.3 mM). Reaction samples were shaken at 30 °C and 800
rpm for 24 hours. The procedure was adapted according to
the parameters investigated (e.g. enzyme amount, buffer
composition and pH, temperature, co-solvent addition etc.).
Biotransformations
for
NMR
analytics.
a) Reaction conditions: substrate (10 mM), methyl acceptor
2c (50 mM), MTase I (freeze-dried CFE, 40 mg mL-1), CP
(400 µL mL-1 reconstituted solution), organic co-solvent
(10% v/v, as indicated) in MOPS/KOH buffer (50 mM, pH
Biotransformations were started on 1 mL scale as
described above 32 times in parallel. After 24 h incubation,
the biotransformation was centrifuged for 10 min at 14680
rpm. All supernatants were pooled in a centricon and
basified to pH 8.0 with a saturated aqueous NaHCO3
solution. The aqueous phase was extracted with EtOAc (5
x 15 mL). The phases were separated by centrifugation at
9000 rpm for 20 min. The combined organic phase was
dried (Na2SO4) and the solvent was evaporated under
reduced pressure. The products of the methyl donors 1a, p-
1d and 2,3-1e were purified via preparative HPLC (1a, p-
1d Method A, see table S2; 2,3-1e Method B, see table S2).
(In cases using 2b as acceptor, excess of 2b may be
removed by SPE; not performed here). Catechol 2a (10 mg,
28%), 3,4-dihydroxybenzyl alcohol 2d (9.4 mg, 19%) and
3-methoxycatechol 2e (6 mg, 12%) were isolated by this
procedure.
b)
6.5, 150 mM KCl) at 30 °C, 800 rpm for 24 h. Reaction
conditions as in a) but with methyl acceptor 2b.
c)
Conversions determined after 24 h by HPLC-UV from
areas for substrate and demethylated product using
calibration curves. Deviation values (s.e.m., nꢀ=ꢀ3) lie
between 0.1 and 3.3 % for the substrates tested.
Experimental Section
All chemicals and solvents were obtained from commercial
suppliers (Sigma Aldrich/Fluka, VWR International/Merck,
Roth) and used as such unless stated otherwise. Cloning of
genes and recombinant expression in E. coli was
Determination of conversions. After specified time points
(e.g. 30 min, 1 h, 2 h, 24 h) a reaction aliquot (30 µL) was
withdrawn, quenched by the addition of MeCN (180 µL),
incubated at room temperature (30 min) and diluted with
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
deionized water (90 µL). Precipitated protein was removed
by centrifugation (14000 rpm, 15 min) and the clear
supernatant was filtered and directly analysed by HLPC
(Agilent 1260 Infinity system equipped with an UV
detector) using an achiral C18 column (Phenomenex, Luna,
C18 100c, 250x4.6mm, 5mm). H2O/MeCN (0.1% TFA)
was used as eluent with a flow rate of 1 mL/min.
Compounds were detected by UV-absorption and
conversions were calculated based on calibrations curves.
Q. Li, L. F. Chi, Angew. Chem. Int. Ed. 2016, 55, 9881-
9885.
[13] R. R. Burgess, Methods Enzymol. 2009, 463, 259-282.
[14] J. A. Dodge, M. G. Stocksdale, K. J. Fahey, C. D.
Jones, J. Org. Chem. 1995, 60, 739-741.
[15] A. Singh, V. Upadhyay, A. K. Upadhyay, S. M.
Singh, A. K. Panda, Microb. Cell Fact. 2015, 14.
Determination of initial rates. In order to enable a better
comparison of different characterization studies, initial
rates were determined for most conditions investigated.
For this purpose the demethylation of guaiacol was
performed under respective conditions according to the
procedure described above and assayed over the first time
period of 1-2 h (representing the linear range of the
reaction, for examples see Figure S4). The amount of
product formed after specified time points was plotted
against the reaction time and the slope of linear correlation
was used to calculate the initial reaction rate. Enzyme
activity (mU) was defined as the amount of enzyme that
catalyzes the conversion of 1 nanomole of substrate per
minute.
[16] J. M. Hagel, P. J. Facchini, Front. Physiol. 2010, 1.
[17] G. F. White, N. J. Russell, E. C. Tidswell, Microbiol.
Rev. 1996, 60, 216-232.
[18] J. Dong, E. Fernández-Fueyo, F. Hollmann, C. E.
Paul, M. Pesic, S. Schmidt, Y. Wang, S. Younes, W.
Zhang, Angew. Chem. Int. Ed. 2018, 57, 9238-9261.
[19] B. M. Nestl, S. C. Hammer, B. A. Nebel, B. Hauer,
Angew. Chem. Int. Ed. 2014, 53, 3070-3095.
[20] H. Sun, H. Zhang, E. L. Ang, H. Zhao, Bioorg. Med.
Chem. 2018, 26, 1275-1284.
Acknowledgements
[21] M. Bučko, P. Gemeiner, A. Schenkmayerová, T.
Krajčovič, F. Rudroff, M. D. Mihovilovič, Appl.
Microbiol. Biotechnol. 2016, 100, 6585-6599.
This study was financed by the Austrian FFG, BMWFJ, BMVIT,
SFG, Standortagentur Tirol and ZIT through the Austrian FFG-
COMET- Funding Program (Austrian Centre of Industrial
Biotechnology). Financial support by the Austrian Science fund is
gratefully acknowledged (P30920-B21).
[22] X. J. Ji, Y. Z. Li, L. Q. Xie, H. J. Lu, W. Ding, Q.
Zhang, Angew. Chem. Int. Ed. 2016, 55, 11845-11848.
[23] M. Node, H. Hori, E. Fujita, J. Chem. Soc., Perkin
Trans. I 1976, 2237-2240.
References
[24] J. M. Hagel, P. J. Facchini, Nat. Chem. Biol. 2010, 6,
[1] R. L. Burwell, Chem. Rev. 1954, 54, 615-685.
273-275.
[2] S. E. Hennig, S. Goetzl, J. H. Jeoung, M. Bommer, F.
Lendzian, P. Hildebrandt, H. Dobbek, Nat. Commun.
2014, 5.
[25] V. Snieckus, Chem. Rev. 1990, 90, 879-933.
[26] D. A. Evans, M. D. Ennis, D. J. Mathre, J. Am. Chem.
Soc. 1982, 104, 1737-1739.
[3] B. P. Callahan, R. Wolfenden, J. Am. Chem. Soc. 2003,
125, 310-311.
[27] L. Reisky, H. C. Büchsenschütz, J. Engel, T. Song, T.
Schweder, J.-H. Hehemann, U. T. Bornscheuer, Nat.
Chem. Biol. 2018, 14, 342-344.
[4] P. Lorenzo-Luis, A. Romerosa, M. Serrano-Ruiz, ACS
Catal. 2012, 2, 1079-1086.
[28] T. Oku, Y. Arita, H. Tsuneki, T. Ikariya, J. Am. Chem.
Soc. 2004, 126, 7368-7377.
[5] A. Fredriksson, S. Stone-Elander, J. Label. Compd.
Rad. 2002, 45, 529-538.
[29] F. S. Mingo, S. Studenik, G. Diekert, FEMS
Microbiol. Ecol. 2014, 90, 783-790.
[6] G. R. Pettit, H. Hoffmann, D. L. Herald, J. McNulty, A.
Murphy, K. C. Higgs, E. Hamel, N. E. Lewin, L. V.
Pearce, P. M. Blumberg, R. K. Pettit, J. C. Knight, J.
Org. Chem. 2004, 69, 2251-2256.
[30] A. Schilhabel, S. Studenik, M. Vodisch, S. Kreher, B.
Schlott, A. Y. Pierik, G. Diekert, J. Bacteriol. 2009,
191, 588-599.
[7] W. E. S. Hart, L. Aldous, J. B. Harper, Org. Biomol.
Chem. 2017, 15, 5556-5563.
[31] E. Rosini, P. D'Arrigo, L. Pollegioni, Catal. Sci.
Technol. 2016, 6, 7729-7737.
[8] J. F. W. McOmie, M. L. Watts, D. E. West,
Tetrahedron 1968, 24, 2289-2292.
[32] C. Zhang, X. Sun, S. H. Xu, B. Y. Yu, J. Zhang, Appl.
Biochem. Biotechnol. 2017, 183, 1026-1034.
[9] I. Ryu, H. Matsubara, S. Yasuda, H. Nakamura, D. P.
Curran, J. Am. Chem. Soc. 2002, 124, 12946-12947.
[33] E. Lanfranchi, M. Trajkovic, K. Barta, J. G. de Vries,
D. B. Janssen, ChemBioChem 2019, 20, 118-125.
[10] C. Pasquini, A. Coniglio, M. Bassetti, Tetrahedron
Lett. 2012, 53, 6191-6194.
[34] M. R. Bennett, S. A. Shepherd, V. A. Cronin, J.
Micklefield, Curr. Opin. Chem. Biol. 2017, 37, 97-106.
[11] J. Cornella, C. Zarate, R. Martin, Chem. Soc. Rev.
2014, 43, 8081-8097.
[35] S. Mordhorst, J. Siegrist, M. Muller, M. Richter, J. N.
Andexer, Angew. Chem. Int. Ed. 2017, 56, 4037-4041.
[12] B. Yang, H. P. Lin, K. J. Miao, P. Zhu, L. B. Liang,
K. W. Sun, H. M. Zhang, J. Fan, V. Meunier, Y. Y. Li,
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
[36] C. Sommer-Kamann, A. Fries, S. Mordhorst, J. N.
Andexer, M. Muller, Angew. Chem. Int. Ed. 2017, 56,
4033-4036.
[44] H. Sjuts, M. S. Dunstan, K. Fisher, D. Leys, Acta.
Crystallogr. D Struct. Biol. 2013, 69, 1609-1616.
[45] C. L. Drennan, S. Huang, J. T. Drummond, R. G.
Matthews, M. L. Ludwig, Science 1994, 266, 1669-
1674.
[37] N. Richter, F. Zepeck, W. Kroutil, Trends Biotechnol.
2015, 33, 371-373.
[38] R. G. Matthews, M. Koutmos, S. Datta, Curr. Opin.
Struct. Biol. 2008, 18, 658-666.
[46] D. J. Ferguson, Jr., D. G. Longstaff, J. A. Krzycki,
Methods Enzymol. 2011, 494, 139-158.
[39] J. E. Farnberger, N. Richter, K. Hiebler, S.
Bierbaumer, M. Pickl, W. Skibar, F. Zepeck, W.
Kroutil, Commun. Chem. 2018, 1, 82.
[47] A. P. Green, N. J. Turner, Perspect. Sci. 2016, 9, 42-
48.
[48] N. J. Turner, E. O'Reilly, Nat. Chem. Biol. 2013, 9,
[40] J. R. Roth, J. G. Lawrence, T. A. Bobik, Annu. Rev.
Microbiol. 1996, 50, 137-181.
285-288.
[49] R. de Souza, L. S. M. Miranda, U. T. Bornscheuer,
[41] J. Bridwell-Rabb, C. L. Drennan, Curr. Opin. Chem.
Biol. 2017, 37, 63-70.
Chem. Eur. J. 2017, 23, 12040-12063.
[50] N. J. Turner, L. Humphreys, Biocatalysis in Organic
Synthesis: The Retrosynthesis Approach, Royal Society
of Chemistry, 2018.
[42] K. Gruber, B. Puffer, B. Kräutler, Chem. Soc. Rev.
2011, 40, 4346-4363.
[43] S. Studenik, M. Vogel, G. Diekert, J. Bacteriol. 2012,
194, 3317-3326.
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801590
Advanced Synthesis & Catalysis
UPDATE
Biocatalytic Methyl Ether Cleavage:
Characterization of the Corrinoid-Dependent
Methyl Transfer System from Desulfitobacterium
hafniense
Adv. Synth. Catal. Year, Volume, Page – Page
Nina Richter, Judith E. Farnberger, Simona
Pompei, Christopher Grimm, Wolfgang Skibar,
Ferdinand Zepeck, and Wolfgang Kroutil*
9
This article is protected by copyright. All rights reserved.