ACS Catalysis
Research Article
methyl group from the cofactor methylcobalamin (Me-
In a next step, various pairs of methyl donors and acceptors
were analyzed to get a first idea of the substrate scope.
Thereby, the 1,3-phenyldiol orcinol 4n unexpectedly stood out
see Supporting Information, Figure S7). This was a surprise
since the substitution pattern is not related to veratrol or its
demethylated product guaiacol 2a. Orcinol 4n was methylated
to 3-methoxy-5-methylphenol 2n. Furthermore, 3,4-dimethox-
ytoluene 1b differing to veratrol 1a by one methyl group, was
demethylated with better conversion compared to veratrol
(Scheme 1).
III 41−44
Cob )
to the substrate giving cobalt in the oxidation
I
state I (Cob ). Methylcobalamin was then regenerated by the
same MTase using a methyl donor like guaiacol 2a as
cosubstrate. Thus, for an overall methyl transfer reaction, a
methyl donor and a methyl acceptor are required. By using an
excess of one compound, the reaction may be shifted toward
demethylation or methylation. It is worthwhile to note that the
cobalamin is bound to a carrier protein, the corrinoid protein
(
CP), which was in the case above the corrinoid protein
dhaf4611 originating from the same organism.
Unfortunately, the MTase from D. hafniense is limited to
guaiacol derivatives as substrates for demethylation (i.e.,
methyl donor) and catechol derivatives as methyl acceptor.
Thus, in a typical reaction, the methyl transfer occurred from
the substrate guaiacol 2a as methyl donor to 3,4-dihydrox-
The methyltransfer reaction employing MT-vdmB/
dhaf4611 was optimized regarding the type of buffer salt,
2
+
pH, the concentration of Zn , temperature, cosolvents, and
the ratio between MT-vdmB and the carrier protein dhaf4611.
Best conversions were obtained at pH 6.5 in 50 mM HEPES,
MES, or MOPS buffer (Supporting Information, Figure S1).
Nevertheless, the biocatalyst also tolerated basic conditions in
CHES buffer (pH 9.5 and 10, Supporting Information, Figure
31,34,35
ybenzoic acid 4c as methyl acceptor.
Since many natural and pharmaceutical compounds
45,46
contain more than one protected methoxy group in close
proximity and due to the limitation of the substrate pattern of
the MTase from D. hafniense, alternative enzymes transforming
substrates with fewer restrictions are needed.
47
2+
S2). According to literature, the MT-vdmB is probably Zn
dependent due to a unique zinc-binding motif D-X -C-X -C.
2
7
39
Indeed, the highest conversion was obtained in the presence of
0 μM of ZnCl , which was about twice as much as in the
2
2
RESULTS AND DISCUSSION
2+
2+
■
absence of Zn ; higher concentrations (≥50 μM Zn ) led to
less conversion (Supporting Information, Figure S3). Fur-
thermore, the optimal temperature was found to be 35 °C
As a starting point, we chose the methyltransferase veratrol-O-
32,47−49
demethylase (MT-vdmB)
and the carrier protein vdmA
both originating from the same organism, namely the
50,51
anaerobic bacterium Acetobacterium dehalogenans.
The
To improve the bioavailability of less water-soluble
substrates in buffer, DMSO was investigated as cosolvent at
different concentrations (0−10%). Interestingly, best con-
versions were obtained without addition of cosolvent (18%
conv.) when using substrate 1b (10 mM) and methyl acceptor
4n (20 mM); although solubility is not an issue for these
compounds at the concentration used, 2% v/v DMSO was
substrates (17% conv., Supporting Information, Figure S5). In
comparison to other cosolvents such as MeOH, EtOH, 1,4-
dioxane and THF, DMSO performed the best (Supporting
Information, Figure S6). Finally, the optimal ratio of the
methyltransferase and the carrier protein was investigated.
Applying a 1:20 ratio of the MT-vdmB/carrier protein led to
highest conversion (30% conv., Supporting Information, Table
methyltransferase MT-vdmB is described to demethylate
veratrol 1a to guaiacol 2a (Scheme 1); thus, here two methoxy
Substrates 1a and 1b
Subsequently, the optimized conditions were applied for the
demethylation of veratrol 1a and a range of 1,2-dimethoxy
substituted substrates 1b-f using a 2-fold molar excess of
orcinol 4n as methyl acceptor. At these conditions, veratrol 1a
was preferentially monodemethylated to guaiacol 2a (54%
conv.), giving at this stage of the reaction a tiny amount of the
didemethylated product catechol 4a (1%, Table 1, entry 1).
Veratrol derivatives were efficiently demethylated with up to
77% conversion as observed for the 4-methyl substituted
substrate 1b (Table 1, entries 2−6).
For selected substrates mono- as well as didemethylation
was observed, like for 1a-c and 1f. On the other hand, for
substrates 1d and 1e exclusively monodemethylated products
were observed at the analyzed stage of conversion; to perform
exclusively monodemethylation is challenging if not impossible
by chemically means. Especially 3,4-dimethoxyphenol 1d is
worthwhile to mention, since the monodemethylation
occurred with perfect regioselectivity, namely in meta-position
to the phenolic OH of the substrate 1d leading exclusively to
4-methoxyresorcinol 3d with >99% regioselectivity. So far,
groups are present in 1,2-position. This type of substrate was
not accepted by the previously described MTase from D.
3
1,34−36
hafniense.
However, when MT-vdmB was used in
combination with the carrier protein vdmA from the same
organism as described in literature, only very low conversion
for demethylation of veratrol 1a (10 mM substrate
concentration) was observed (3%) when using a 5-fold
molar excess of 3,4-dihydroxybenzaldehyde 4c as a methyl
acceptor (Scheme 1). The acceptor was converted to the
regioisomers vanillin 2c and isovanillin 3c in a 3:1 ratio. Since
optimization studies did not improve the conversion, an
alternative carrier protein was considered. Aligning carrier
proteins from other hosts with vdmA, the carrier protein
dhaf4611 from Desulfitobacterium hafniense showed the highest
sequence identity with 72% (EMBOSS needle). Remarkably,
testing now the combination of MT-vdmB with dhaf4611 led
to a higher conversion of veratrol 1a (10% conv.) compared
with the reaction with the “natural” carrier protein vdmA from
the same organism (3% conv.).
1
0376
ACS Catal. 2020, 10, 10375−10380