Biosynthesis of branched alkoxy groups: Iterative methyl group alkylation by a cobalamin-dependent radical SAM enzyme

Article abstract of DOI:10.1021/jacs.6b10901

The biosynthesis of branched alkoxy groups, such as the unique t-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metab

Full text of DOI:10.1021/jacs.6b10901

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Communication
Biosynthesis of branched alkoxy groups: iterative methyl group
alkylation by a Cobalamin-Dependent Radical SAM Enzyme
Yuanyou Wang, Bastien Schnell, Sascha Baumann, Rolf Müller, and Tadhg P. Begley
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b10901 • Publication Date (Web): 31 Dec 2016
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Biosynthesis of branched alkoxy groups: iterative methyl group al-
kylation by a Cobalamin-Dependent Radical SAM Enzyme
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Yuanyou Wang†, Bastien Schnell‡, Sascha Baumann‡, Rolf Müller‡ and Tadhg P. Begley†*
†Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
‡Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Center
for Infection Research, Saarland University, Universitätscampus E8.1, D-66123 Saarbrücken, Germany
Supporting Information Placeholder
ABSTRACT: The biosynthesis of branched alkoxy groups like
the unique t-butyl group found in a variety of natural products, is
CO2H
A
CO2H
H
still poorly understood. Recently, cystobactamids were isolated
and identified from Cystobacter sp as novel antibacterials. These
HO2C
HO2C
H3C SR2
metabolites contain an isopropyl group proposed to be formed
using CysS, a cobalamin-dependent radical S-adenosylmethionine
1
O
(SAM) methyltransferase. Here, we reconstitute the CysS-
catalyzed reaction and demonstrate that it not only performs
sequential methylations of a methyl group to form isopropyl
groups but remarkably also t-butyl groups on p-aminobenzoate
CO2H
HO
O
O
O
O
HO2C
OH
O
thioester substrates. To our knowledge, this is the first in vitro
O
reconstitution of a cobalamin-dependent radical SAM enzyme
2
catalyzing the conversion of a methyl group to a t-butyl group.
Ginkgolides
B
H
H
H
Natural products with branched alkoxy groups play an im-
portant role in the development of bioactive compounds. In par-
ticular, the t-butyl group has fascinated organic chemists for more
than a century and has played a major role in mechanistic studies
on organic substitution reactions and the design and characteriza-
tion of theoretically interesting molecules such as the remarkable
tetra t-butyl tetrahedrane.1 While numerous branched alkoxy
group substituted terpenes, polyketides and peptides have been
identified, experimental studies on the biosynthesis of t-butyl
groups are still at an early stage and many of the mechanistic
proposals in the literature have not been adequately experimental-
ly tested.2
O
O
O
O
O
O
O
O
O
5
4
3
H
O
OH
Oxidation
O
O
O
O
O
O
O
O
O
8
7
6
Swietenone
S
S
IcmF
CoA
CoA
C
For the ginkgolides and several other t-butyl substituted ter-
penes, the t-butyl group is formed by a double bond methylation
using S-adenosylmethionine (Figure 1A).3 Formation of the t-
butyl group in the coumarin swietenone is proposed to involve
carbocation insertion into a CH bond to give a cyclopropyl inter-
mediate, which then undergoes acid mediated ring-opening (Fig-
ure 1B).4 The biosynthesis of pivalic acid, a starter unit in the
biosynthesis of t-butyl substituted polyketides, is mediated by a
vitamin B12-dependent enzyme (Figure 1C).5-6 Very recently, the
B12/radical SAM mediated conversion of isopropyl glycine to t-
butyl glycine in the polytheoamide propeptide was reported (Fig-
ure 1D).7-8 The latter two enzymes are the only t-butyl biosynthe-
sis enzymes that have been experimentally reconstituted.
O
O
Ado-CH3
Ado-CH2
10
9
S
S
CoA
CoA
O
O
11
12
D
H
N
H
PoyC
R
R
N
N
H
R'
N
R'
H
O
O
14
13
Figure 1. Mechanistic proposals for the formation of the t-butyl
group in representative natural products.
Radical SAM enzymes use the 5¢-deoxyadenosyl radical (5¢-
dA•), generated by reductive cleavage of SAM, to initiate a di-
verse set of radical reactions.9 A subfamily of these enzymes
combines adenosyl radical chemistry with methyl cobalamin
chemistry enabling the methylation of non-nucleophilic centers in
natural product biosynthesis.10
Cobalamin-dependent radical SAM methyltransferases are ex-
perimentally challenging and are generally difficult to overpro-
duce. Only a few systems have been reconstituted.11 These in-
clude enzymes that catalyze phosphinic acid methylation (PhpK,
L-phosphinothricin biosynthesis),12 alcohol C-methylation (GenK,
gentamicin biosynthesis13 and Fom3, fosfomycin biosynthesis14),
iterative C-methylation to form the ethyl group (ThnK, car-
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bapenem biosynthesis)15 and indole C-methylation (TsrM, thio-
mer of CysS, demonstrating partial cluster formation in the over-
expressed protein.
strepton biosynthesis).16
1
2
3
4
5
6
7
8
9
The cystobactamids 17 are a novel class of isopropyl substitut-
ed antibacterial compounds produced by myxobacteria.17 The
biosynthetic gene cluster has been identified and sequence analy-
sis suggested that CysS is a cobalamin-dependent radical SAM
methyltransferase, potentially involved in the iterative methyla-
tion of the 3-methoxy-4-aminobenzoic acid moieties of cystobac-
tamid 15 (Figure 2). Some minor derivatives exhibit methyl,
ethyl, isopropyl and sec-butyl groups (Stephan Hüttel and R.M.,
unpublished results) supporting the hypothesis of CysS being an
enzyme iteratively adding methyl-groups to its substrate.
Several p-aminobenzoic acid (PABA) analogs were tested as
substrates for CysS (Table S1). None gave the desired methylated
product as indicated by LC-MS analysis. Further analysis of the
cystobactamid biosynthesis cluster suggested the coenzyme A or
the acyl carrier protein thioester of 15 (CysG)17 as possible CysS
substrates. To test this proposal, N-acetylcysteamine thioester 18
was synthesized and incubated with CysS, SAM, MeCbl, and
flavodoxin/flavodoxin reductase/NADPH (Figure 4). LC-MS
analysis of the resulting reaction mixture demonstrated the for-
mation of the ethyl ether 19. This was further confirmed by co-
elution of the reaction product with a synthesized sample of 19
(Figure S2). When the ethyl ether 19 was incubated with CysS,
the isopropyl ether 20 was detected by LC-MS analysis (Figure
S2).
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O
OH
OCH3
H2NOC
O
O
H2N
15
a: R = Et
b: R = i-Pro
c: R = sec-Bu
O
NH HN
Pantetheinyl thioester 21 was a better substrate for CysS and
iterative methylations to give the ethyl, isopropyl and the butyl
ethers were detected by LC-MS analysis (Figure 5). Small
amounts of the ethers 22a and 22b were detected in the absence of
the reducing agent suggesting that some of the purified enzyme
contained the reduced [4Fe-4S] cluster. To confirm the structures
of 22a-c, authentic samples of these compounds were synthesized.
The enzymatic products matched the synthetic standards in terms
of retention time, exact mass, and fragmentation pattern (Figure 5,
Figure S3-5). In addition, CysS catalyzed the conversion of syn-
thetic 22a to 22b-d and the conversion of synthetic 22b to 22c, d
(Figure S6). The second component in the extracted ion chroma-
togram for the t-butyl ether 22c (Figure 5C and D) was identified
as the sec-butyl ether 22d by co-migration with an authentic
standard of 22d (Figure S7).
O
CysS
HN
O
O
NH
O
O
R
OH
OH HN
H2N
O
17
O
R
COOH
R
16
NO2
Figure 2. Proposed formation of the branched alkoxy groups of
cystobactamids by CysS-catalyzed iterative methylations of a
methyl ether.
To test this hypothesis, an in vivo labeling experiment using
[13C-Methyl]-L-methionine was performed to determine the origin
of the isopropyl groups on cystobactamid 919-1 (17b). LC-MS
analysis of the extracts showed a mass shift of +7 m/z indicating
that the seven carbons from the methoxy and both isopropyl
groups were from methionine and therefore most likely SAM
derived (Figure 3).
O
O
O
A
O
S
O
S
O
S
N
N
N
H
H
H
CysS SAM MeCbl
Reducing system
CysS SAM MeCbl
Reducing system
O
O
O
NH2
NH2
NH2
18
19
20
O
O
O
O
B
O
S
OH
O
S
OH
N
N
N
N
CysS SAM MeCbl
Reducing system
H
H
H
H
OH
OH
R
a: R = Et
O
O
b: R = i-Pro
c: R = t-Bu
d: R = sec-Bu
NH2
NH2
22
21
Figure 4. Identification of two substrates for CysS. A competition
reaction with a 1:1 mixture demonstrated that 21 is 47 times more
reactive than 18 (Supporting information).
Figure 3. MS analysis of cell extracts containing (A) methionine
and (B) [13C-Methyl]-L-methionine showing the incorporation of
up to seven 13C in cystobactamid 919-1.
Various [4Fe-4S] cluster reducing agents were tested in addi-
tion
to
the
flavodoxin/flavodoxin
reductase/NADPH.
Here we describe the successful in vitro reconstitution of
CysS and demonstrate that this enzyme can assemble isopropyl,
sec-butyl and t-butyl groups by sequential methylations of a me-
thyl group. To our knowledge, this is the first example of an
isopropyl, sec-butyl and a t-butyl group biosynthesis from a me-
thyl group using radical chemistry. Sequence analysis suggests
that related chemistry is involved in the biosynthesis of other
natural products such as SW-163G18 and bottromycin.19-20
NADPH/methyl viologen, a commonly used electron source for
cobalamin-dependent radical SAM enzymes, gave similar activi-
ty.13, 15 However, dithionite or the combination of methyl viologen
and dithionite gave a significantly lower activity.22 Buffer thiols
inactivate the substrate by trans thioesterification and need to be
avoided.
Quantitative analysis of the enzymatic reaction mixture (CysS,
methyl ether 21, flavodoxin/flavodoxin reductase/NADPH, reac-
tion run to completion) by LC-MS showed that 1 equivalent of
enzyme undergoes >2 turnovers, generating around 2.0 equiva-
lents of 5¢-dA, 2.0 equivalents of SAH, 1.4 equivalents of ethyl
ether 22a and 0.3 equivalents of isopropyl ether 22b (Figures 5
and 6, SI). The t-butyl ether 22c was detected only when the
concentration of the isopropyl ether 22b was >23 µM. The ratio of
5¢-dA to SAH was close to 1, suggesting that two molecules SAM
were consumed for each methylation reaction and that the uncou-
pled production of 5¢-dA is low. This is consistent with SAM
CysS was cloned into a pET28b vector and co-expressed with
a plasmid encoding the suf operon ([4Fe-4S] biosynthesis)21 in
E.coli BL21 (λDE3). The protein was then purified, under anaer-
obic conditions, by Ni-NTA affinity chromatography. Cobalamin
was not required in the growth medium for production of soluble
protein. The UV-visible spectrum of purified CysS revealed a
420-nm shoulder, typical of a bound Fe/S cluster (Figure S1). Iron
and sulfide analysis yielded 2.5 irons and 2.8 sulfides per mono-
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functioning as the source of both the adenosyl radical and the
thus allowing for the formation of higher levels of the t-butyl
ether. Our studies on CysS suggest that t-butyl substituted cysto-
bactamids, which have not yet been isolated, are likely to exist.
1
2
3
4
methyl group and was further supported by LC-MS analysis of a
reaction mixture containing CD3-SAM which demonstrated CD3
incorporation into the ethyl ether 22a and the isopropyl ether 22b
(Figure 7).
A
NH2
N
N
5
6
7
N
N
A
O
O
O
O
S
OH
5’dA
N
H
N
H
OH OH
OH
O
8
9
22a
NH2
NH2
N
B
N
N
5
10
11
12
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19
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21
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N
HOOC
S
B
O
O
O
NH2
SAH
O
S
OH
N
H
N
H
OH OH
OH
O
22b
NH2
5
10
15
20
25
30
35
Retention time (minutes)
C
O
O
Figure 6. LC-MS detection of 5¢-dA and SAH in the CysS-
catalyzed iterative methylations of the methyl ether 21. Red trace
is for the complete reaction mixture. Green trace is for reaction
mixtures where the reducing system (flavodoxin/flavodoxin re-
ductase/NADPH) is absent. (A) EICs of 5¢-dA [M+H]+
(252.11±0.02). (B) EICs of SAH [M+H]+ (385.13±0.02). Orange,
green, cyan, and black traces are for reaction mixtures where
either CysS, reducing system, MeCbl or substrate is absent. The
product ratio was determined by calibrating the signal intensity
with known concentrations of standards (SI).
O
S
OH
N
N
H
H
OH
O
22c
NH2
D
O
O
O
S
OH
N
H
N
H
OH
O
22c
NH2
16
20
24
Retention time (minutes)
28
Figure 5. LC-MS analysis of the CysS-catalyzed iterative methyl-
ation of the methyl ether 21. Red trace is for the complete reaction
mixture. Green trace is for reaction mixtures where the reducing
system (flavodoxin/flavodoxin reductase/NADPH) is absent.
Ethyl ether 22a, isopropyl ether 22b were not formed in the con-
trol reactions lacking CysS, SAM or MeCbl. (A)
Extracted Ion
Chromatograms (EICs) of the ethyl ether 22a [M+H]+
(442.20±0.02). (B) EICs of the isopropyl ether 22b [M+H]+
(456.22±0.02). (C) EICs of the t-butyl ether 22c [M+H]+
(470.23±0.02). (D) EICs of [M+H]+ (470.23±0.02) showing co-
migration with a synthesized sample of 22c. Cyan trace is the t-
butyl ether standard. Blue trace is co-elution of the enzymatic
product and synthetic standard. The second component in the
extracted ion chromatogram for the t-butyl ether 22c (panels C
and D) was identified as the sec-butyl ether 22d (Figure S7). The
product ratio was determined by calibrating the signal intensity
with known concentrations of standards (SI).
Figure 7. MS analysis of a reaction mixture in which CH3-SAM is
replaced with CD3-SAM showing CD3 incorporation into the
ethyl and isopropyl ethers of 22a and 22b respectively. Panels A
and C: Mass spectra of 22a and 22b formed from CH3-SAM.
Panels B and D: Mass spectra of 22a and 22b formed from CD3-
SAM.
A mechanistic proposal for the CysS-catalyzed reaction, based
on the proposed mechanisms for GenK13 and ThnK15, is shown in
Figure 8. After initial formation of methylcobalamin by SAM
mediated methylation, reductive cleavage of SAM by the [4Fe-
4S]+1 cluster generates the 5¢-deoxyadenosyl radical. This ab-
stracts a hydrogen atom from the methyl group of the substrate 21
to give radical 23, which then undergoes a radical substitution
with methyl cobalamin to give the ethyl ether 22a. An analogous
methyl transfer, by a radical substitution mechanism, has prece-
dence in cobalamin model chemistry.23 Regeneration of MeCbl
from Cbl(II) can be achieved by reduction to Cbl(I) by the [4Fe-
4S]+1 cluster followed by SAM-mediated methylation. Repetition
of this sequence results in the successive formation of the isopro-
pyl, t-butyl and sec-butyl ethers of 21. The in vitro ratio of
branched alkoxy groups is likely to be different from the in vivo
ratio because in vivo each methylation in the iterative sequence is
in competition with the next step in the biosynthesis. This is not
the case for the purified enzyme where no such competition exists
In summary, we have elucidated the enzymology of a radical-
mediated conversion of a methyl ether to a t-butyl ether. CysS is a
cobalamin dependent radical SAM methyltransferase that catalyz-
es the iterative methylation of a substrate methyl ether to give
ethyl, isopropyl, t-butyl and sec-butyl substituted products. Each
methyl transfer is likely to proceed via hydrogen atom abstraction
from the evolving carbon of the substrate followed by a radical
substitution on methyl cobalamin. This biosynthetic strategy in
principle enables the host myxobacterium to biosynthesize a
combinatorial antibiotic library of 25 cystobactamid analogs. The
analysis of the impact of these molecular decorations on bioactivi-
ty will be the task of future studies.
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Co(III)
CH3
Co(I)
O
SR
21,e-
O
SR
Co(III)
CH3
Co(III)
CH3
H
Met
Me
S
H
C
S
Ade
Ade
H
H
Me
R'
R'
C
H
H
O
O
O
O
21
SAH
21
NH2
NH2
OH OH
SAH
OH OH
SAM
S
Ade
CH2
Ade
R'
[4Fe-4S]1+
O
O
[4Fe-4S]2+
OH OH
5'-dA
[4Fe-4S]2+
[4Fe-4S]2+
OH OH
SAM
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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O
SR
O
SR
Co(III)
CH3
SAM, e-
Co(II)
Co(II)
Me
Co(I)
CH3
H
H
Me
S
H
S
C
Ade
O
Ade
R'
O
H
R'
O
O
23
22a
NH2
22a, 5'dA
NH2
CH3
Ade
CH3
O
Ade
OH OH
SAM
OH OH
SAM
O
OH OH
5'-dA
[4Fe-4S]2+
OH OH
5'-dA
[4Fe-4S]1+
[4Fe-4S]2+
[4Fe-4S]2+
O
SR
O
SR
Co(III)
CH3
Co(III)
O
SR
CH3
22a,SAM, e-
SAH
CH3
CH3
H
CH3
CH3
CH3
CH3
S
H
H
Ade
R'
O
O
O
O
22b
R:
22c
22a
NH2
NH2
Me
S
NH2
OH OH
SAH
Ade
R'
O
[4Fe-4S]1+
[4Fe-4S]2+
O
O
OH OH
SAM
COOH
NH2
R':
S
OH
N
N
H
H
OH
Figure 8. Mechanistic proposal for CysS-catalyzed iterative methylations to form branched alkoxy groups. The mechanism assumes two
different SAM binding sites. It is also possible that SAM binds to a single site and that the position of the sulfonium moiety is altered by a
protein conformational change.
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ASSOCIATED CONTENT
Supporting Information
Experimental details regarding the labeling experiments, the
expression and purification of CysS, the syntheses of substrates,
NMR spectra and LC-MS analysis are described in the supporting
information which is available, free of charge, via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*begley@chem.tamu.edu
ORCID
Tadhg P. Begley: 0000-0001-5134-2623
ACKNOWLEDGMENT
This work was supported by NIH (DK44083) and by the
Robert A. Welch Foundation (A-0034) as well as the German
Centre for Infection Research (DZIF).
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Table of Contents
9
O
SR'
O
SR'
O
SR'
O
SR'
Co(II)
Co(III)
CH3
Co(III)
CH3
Met
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH3
H
H
CH3
R
CH2
O
O
O
O
NH2
R'
NH2
NH2
NH2
Me
CH3
Ade
CH3
Ade
S
O
Et
Ade
O
O
i-Pro
t-Bu
sec-Bu
R =
OH OH
OH OH
OH OH
[4Fe-4S]2+
[4Fe-4S]1+
[4Fe-4S]2+
5
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