Radical-mediated enzymatic carbon chain fragmentation-recombination

Article abstract of DOI:10.1038/nchembio.512

The radical S-adenosylmethionine (S-AdoMet) superfamily contains thousands of proteins that catalyze highly diverse conversions, most of which are poorly understood, owing to a lack of information regarding chemical products and radical-dependent transformations. We here report that NosL, involved in forming the indole side ring of the thiopeptide nosiheptide (NOS), is a radical S-AdoMet 3-methyl-2-indolic acid (MIA) synthase. NosL catalyzed an unprecedented carbon chain reconstitution of L-tryptophan to give MIA, showing removal of the C' ±-N unit and shift of the carboxylate to the indole ring. Dissection of the enzymatic process upon the identification of products and a putative glycyl intermediate uncovered a radical-mediated, unusual fragmentation- recombination reaction. This finding unveiled a key step in radical S-AdoMet enzyme-catalyzed structural rearrangements during complex biotransformations. Additionally, NosL tolerated fluorinated L-tryptophan as the substrate, allowing for production of a regiospecifically halogenated thiopeptide that has not been found among the more than 80 members of the naturally occurring thiopeptide family.

Full text of DOI:10.1038/nchembio.512

article
published online: 16 January 2011 | doi: 10.1038/nchembio.512
radical-mediated enzymatic carbon chain
fragmentation-recombination
Qi Zhang1, yuxue li2, dandan chen1, yi yu1, lian duan1, ben shen3 & Wen liu1*
The radical S-adenosylmethionine (S-AdoMet) superfamily contains thousands of proteins that catalyze highly diverse
conversions, most of which are poorly understood, owing to a lack of information regarding chemical products and
radical-dependent transformations. We here report that NosL, involved in forming the indole side ring of the thiopeptide
nosiheptide (NOS), is a radical S-AdoMet 3-methyl-2-indolic acid (MIA) synthase. NosL catalyzed an unprecedented carbon
chain reconstitution of L-tryptophan to give MIA, showing removal of the Ca-N unit and shift of the carboxylate to the indole
ring. Dissection of the enzymatic process upon the identification of products and a putative glycyl intermediate uncovered a
radical-mediated, unusual fragmentation-recombination reaction. This finding unveiled a key step in radical S-AdoMet enzyme–
catalyzed structural rearrangements during complex biotransformations. Additionally, NosL tolerated fluorinated L-tryptophan
as the substrate, allowing for production of a regiospecifically halogenated thiopeptide that has not been found among the more
than 80 members of the naturally occurring thiopeptide family.
hiopeptides are a class of clinically interesting and highly modi- with nosL’s counterpart nocL from nocathiacin (a naturally occur-
fied polythiazolyl antibiotics1–3. The potent activity of many ring analog of NOS) biosynthesis. However, whether NosL alone
T
members against various drug-resistant bacterial pathogens catalyzes such a complex conversion remains unknown, given
has promoted extensive investigations by chemical modification that 1 biosynthesis from L-tryptophan requires multiple chemi-
into new analog development to overcome their physical drawbacks, cally unusual reactions to process the carbon side chain (including
such as water solubility, for clinical use. Thiopeptides possess a removal of the Cα-N unit and shift of the carboxylate to the indole
characteristic macrocyclic core, consisting of a nitrogen-containing ring; Fig. 1b).
6-membered ring central to multiple thiazoles and dehydroamino
In this study, we found that NosL is indeed a radical S-AdoMet
acids but vary in side chains (and/or rings), which append addi- protein that converts L-tryptophan to 1, a process that involves a
tional functionalities (Fig. 1a). In contrast to the heroic efforts radical-mediated, unusual fragmentation-recombination reaction.
required for chemical synthesis4, the newly established biosynthetic Our characterization of NosL took advantage of efficient 1 produc-
pathways for thiopeptide framework formation are remarkably tion in the nosL-expressing Escherichia coli strain and in vivo and/or
concise5–11, showing conserved post-translational modifications on in vitro determination of substrate(s), products (including shunt
a ribosomally synthesized precursor peptide. The reactions include products) and a putative glycyl intermediate in NosL-catalyzed
cyclodehydrations and subsequent dehydrogenations to form aro- conversion. In light of the substrate tolerance of NosL, regiospecific
matic thiazoles, dehydrations to generate dehydroamino acids and fluorination of NOS was achieved via a modified MIA intermediate,
an intramolecular cyclization to afford the nitrogen heterocycle.
yielding a new halogenated thiopeptide that has not yet been found
For most polycyclic thiopeptides, the side ring formations are in the family of naturally occurring thiopeptides.
independent of precursor peptide12–16. They exclusively exploit
L-tryptophan to furnish the variable functional groups, as exempli- RESULTS
fied by the indolic acid moiety of nosiheptide (NOS) and the qui- In vivo validation of NosL as a MIA synthase
nalic acid moiety of thiostrepton (Fig. 1b). Whereas the quinalic Considering that radical S-AdoMet proteins are sensitive to oxygen
acid formation may require seven enzymes to convert L-tryptophan, in in vitro studies17, we first set out to investigate the NosL func-
in a process including methylation, deamination, oxidation, ring tion in vivo (Fig. 2). nosL was introduced to and expressed in
opening and recyclization, reduction and epoxidation6,7, the indolic E. coli BL21(DE3), yielding the recombinant strain SL4101 to pro-
acid moiety formation in NOS biosynthesis involves the products duce NosL in an N-terminally 6-His–tagged form. HPLC analysis
of two genes, nosL and nosN, which encode the putative radical revealed a compound produced by SL4101, which was absent in the
S-AdoMet proteins that process L-tryptophan via a completely dif- control strain SL4100 that carried the vector alone (Fig. 2a). The
ferent route9. Characterization of a NOS analog that had an open molecular formula for this compound, C10H9O2N, was established
side ring supported the methylation activity of NosN, which acts by high-resolution ESI-MS (HR-ESI-MS), showing a [M − H]− ion
on the 3-methylindolyl group to furnish the 3,4-dimethylindole at m/z = 174.05605 (174.05615 calculated). Additionally, 1H, 13C and
moiety. This leaves NosL as a candidate to biosynthesize the key selected 2D NMR analyses confirmed it to be 1, indicating that NosL
intermediate, 3-methyl-2-indolic acid (MIA, 1). We have confirmed is a new MIA synthase. To probe the origin of 1, the nosL-expressing
the essentiality of NosL to 1 formation9,11: inactivation of nosL com- cells of SL4101 in LB broth were harvested and inoculated into a
pletely abolished NOS production, which can be restored either by nutrient-insufficient medium and further incubated with indivi-
feeding extraneous 1 into cells or by heterologous complementation dual amino acids for a time-course analysis of 1 production.
1State Key laboratory of Bioorganic and Natural Products chemistry, Shanghai Institute of organic chemistry, chinese academy of Sciences, Shanghai,
china. 2State Key laboratory of organometallic chemistry, Shanghai Institute of organic chemistry, chinese academy of Sciences, Shanghai, china.
3Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-madison, madison, Wisconsin, USa. *e-mail: wliu@mail.sioc.ac.cn
154
nature chemical biology | vol 7 | march 2011 | www.nature.com/naturechemicalbiology

NATURE ChEMICAL bIOLOgy doi: 10.1038/nchembio.512
article
O
O
a
b
H
NH2
COOH
H
N
O
N
N
NH2
O
N
NH2
N
H
O
OH
S
S
N
H
O
H
O
O
S
N
N
N
H
N
S
N
L-Trp
N
NH
N
H
H
NosL?
Seven enzymes
N
O
HN
N
O
S
N
S
O
H
NH
O
HN
O
OH
S
OH
HO
NH
S
H
HO
N
H
O
O
O
NH
NH
COOH
HN
O
O
HN
N
H
S
O
S
H
N
N
N
H
N
H
MIA (1)
O
O
S
OH
N
N
R
O
O
O
OH
HO
OH
R = H, Nosiheptide (NOS);
Thiostrepton
Thiostrepton
NOS
R = F, 5′-fluoro-NOS (27)
Figure 1 | Structures of polycyclic thiopeptides and their side ring formations. (a) NoS, 5′-fluoro-NoS and thiostrepton. (b) Processing of l-tryptophan
to afford the indolic acid moiety of NoS and the quinalic acid moiety of thiostrepton, respectively, via completely different routes.
Only the addition of L-tryptophan sped up 1 formation with the showing 1 production with the yield approximately sevenfold
apparent rate of 150 μg per liter per min in the initial 4 h, whereas higher than that of the isolated enzyme without reconstitution
supplementation with other amino acids such as L-glycine or L-serine (Fig. 2a and Supplementary Fig. 4a). In the reaction mixture
had no substantial effect on the 1-producing rate (Supplementary the chemical reductant dithionite can be replaced with a natural
Results and Supplementary Fig. 1). This validated the idea that reduction system containing flavodoxin, flavodoxin reductase
L-tryptophan serves as the substrate for the production of 1. The and NADPH, leading to ~100% improvement in 1 formation
relatively high yield of 1 (42 ± 5 mg per liter over a 20-h period) (Supplementary Fig. 6). NosL-catalyzed conversion cannot pro-
facilitated its purification and enabled further in vivo experiments ceed in the absence of S-AdoMet (Fig. 2a), whose turnover by
to examine NosL catalysis.
NosL was further confirmed upon identification of the product
5′-deoxyadenosine (AdoH, 2, Fig. 2b). S-AdoMet consumption
was independent of L-tryptophan but was significantly enhanced
NosL as a radical S-AdoMet protein for MIA formation
On the basis of sequence alignment, NosL falls into the ThiH by the addition of L-tryptophan into the reaction mixture (Fig. 2b
radical S-AdoMet subclass (around 20% identity, Supplementary and Supplementary Fig. 4a). Together with the identification of
Fig. 2), possessing a typical CxxxCxxC motif for [4Fe-4S] cluster methionine (discussed below) as the concomitant product origi-
nucleation and a glycine-containing segment involved in S-AdoMet nating from S-AdoMet, we conclude that NosL, whose activity is
binding. We therefore explored the nature of NosL in regard to its S-AdoMet dependent, catalyzes 1 formation from L-tryptophan via
cofactor binding and subsequent chemical transformations. In the a 5′-deoxyadenosyl radical (Ado•)-initiated process.
CxxxCxxC motif, the cysteine residues (corresponding to Cys95,
Cys99 and Cys102) were systematically replaced with alanine. The NosL-catalyzed carbon chain reconstitution on L-tryptophan
constructs were introduced into E. coli BL21(DE3), yielding the To investigate the mechanism of NosL-catalyzed conversion,
recombinant strains SL4103 (for AxxxCxxC mutation of NosL), we focused on the reconstitution of the carbon side chain of
SL4105 (for CxxxAxxC mutation) and SL4107 (for CxxxCxxA L-tryptophan. [1-13C] and [3-13C]-labeled L-tryptophans were fed
mutation) for 1 examination. These replacements completely abol- individually into SL4101, resulting in enriched 13C resonances for
ished 1 production (Supplementary Fig. 3), demonstrating their the 2-carboxylate (at δ 164.0 for product 3) and 3-methyl group
indispensability to NosL activity. The S-AdoMet binding poten- (at δ 8.83 for product 4) of MIA, respectively (Scheme 1 and
tial was also evaluated in a similar way, by replacing Gly142 with Supplementary Fig. 7). Consistent with similar studies carried out
alanine within the conserved GE/D motif. The recombinant strain in the NOS-producing strain Streptomyces actuosus12,13, these find-
SL4109 that expressed mutant NosL (G142A) lost the ability to pro- ings demonstrated that 1 biosynthesis involves an unprecedented
duce 1 (Supplementary Fig. 3), supporting the idea that NosL is a carbon chain rearrangement of the L-tryptophan precursor, show-
S-AdoMet–dependent protein.
ing (i) the intramolecular migration of the carboxylate to C2 of
Next, NosL was purified from SL4101 to homogeneity for the indole ring, (ii) transformation of the methylene carbon to a
assays in vitro. Although the aerobically isolated enzyme was inac- methyl group and (iii) elimination of the α-carbon and its asso-
tive, the activity of the anaerobically isolated NosL was detect- ciated amino group. The feeding of L-[2H8]-tryptophan also sup-
able but extremely low (Supplementary Fig. 4). NosL was then ported this conclusion by allowing the production of [2H6]-MIA (5)
reconstituted under strictly anaerobic conditions, giving the puri- (HR-ESI-MS m/z calculated for C10D6H2O2N = 180.09371, found
fied sample (Supplementary Fig. 5a) a dark brownish color. This 180.09343), and the feeding also excluded the participation of the
protein contained 5.1 ± 0.5 of iron and 5.7 ± 0.3 of sulfide per benzene ring in this conversion (given no change in deuterium
molecule. The UV-visible absorptions of the reconstituted NosL substitutions, Scheme 1). A 0.04-p.p.m. upfield shift (from δ 2.48
are characteristic for [4Fe-4S] binding proteins (Supplementary relative to CH3) of the signal that corresponds to the CHD2 protons
Fig. 5b), showing a A280/A400 ratio of 3.4:1 and an apparent was found in the 1H NMR spectrum of 5 (Supplementary Fig. 8),
shift caused by reducing with sodium dithionite. Further electron ruling out adjacent hydrogen transfer, either from Cα or from
paramagnetic resonance (EPR) analysis at 13 K gave an axial spec- C2 on the indole ring, to provide the third hydrogen atom of the
trum (Supplementary Fig. 5c) showing approximate g values of methyl group. Further, using L-[2H8]-tryptophan as the substrate
2.02 and 1.91. This supported the binding of a [4Fe-4S]+ cluster in an in vitro assay of NosL-catalyzed reactions showed that the
to NosL. Incubation of the reconstituted NosL with L-tryptophan coproduced AdoH (2) (HR-ESI-MS m/z calculated for C10H13N5O3 =
and S-AdoMet was performed in the presence of dithionite, indeed 252.10967, found252.10983)wasnotdeuteriumlabeled. Thisclearly
nature chemical biology | vol 7 | march 2011 | www.nature.com/naturechemicalbiology
155

NATURE ChEMICAL bIOLOgy doi: 10.1038/nchembio.512
article
a
b
S-AdoMet
AdoH (2)
COOH
N
N
H
H
I
1
6
I
In E. coli
II
II
III
III
In vitro
IV
2
4
6
8
10
12
14
V
Time (min)
d
8
10
12
14
16
18
Asparagine-
dansyl
O
S
Time (min)
N
HOOC
O
HN
HOOC
H
N
H
N
O
CH2
c
S
NH
S
N
N
N
COOH
O2
O
N
NO2
O2
N
NO2
22
24
+
+
[M + H] = 309
[M + H] = 383
I
I
8
10
–
–
[M – H] = 253
[M – H] = 209
II
II
III
IV
III
IV
6
8
10
12
Time (min)
14
16
18
6
8
10
12
Time (min)
14
16
Figure 2 | Characterization of NosL-catalyzed reaction. all the examinations were performed at least in duplicate (each had at least two parallel
samples). (a) validation of Nosl as a mIa synthase. In vivo 1 production in the E. coli strains, including Sl4101 harboring nosL (I) and Sl4100 carrying the
vector alone (II), and in vitro conversion of l-tryptophan to 1 and the shunt product 6 in the presence (III) and in the absence (IV) of S-adomet, with the
authentic 6 as a standard (V). (b) consumption of S-adomet given the absence (I) and the presence (II) of l-tryptophan, in contrast to the results when
Nosl is omitted from the reaction mixture (III). (c) Identification of the products with DNP derivatization by adding (I) and by omitting (II) the substrate
l-tryptophan, when authentic 7 (III) and 9 (IV) were subjected to DNP derivatization to generate the control derivatives. (d) Identification of the putative
amino acid products with dansyl chloride derivatization by using asparagine as an internal standard for quantitative analysis. Production in the presence (I)
and in the absence (II) of l-tryptophan while authentic 21 (III) and 23 (IV) were subjected to dansyl derivatization to generate the control derivatives for
qualitative analysis.
indicates that the Ado•-initiated rearrangement of the carbon chain Quantitative analysis of NosL-catalyzed in vitro reaction
does not start with a C-H bond cleavage on L-tryptophan to gene- We chose three representative products, including 1 and 3-methyl-
rate the substrate radical.
indole (6) from L-tryptophan and AdoH (2) from S-AdoMet,
to quantitatively evaluate NosL-catalyzed in vitro conversion
(Supplementary Fig. 4). The yields of the products were enzyme-
Determination of the coproduct and shunt products
To probe the individual steps in 1 formation, we carried out exten- dose dependent, as the increase of the concentration of NosL
sive survey of the product profile in NosL-catalyzed in vitro conver- accordingly improved the production of 1, 6 and 2 (Supplementary
sion. HPLC analysis of the reaction mixture revealed the first shunt Fig. 4a). Their productions at 25 °C were relatively constant in the
product (Fig. 2a), which was deduced to be 3-methylindole (6) presence of 1 mM dithionite, 500 μM L-tryptophan and 1 mM
upon further GC-MS analysis with an authentic compound as S-AdoMet, showing that about 39 ± 3 μM 1, 140 ± 20 μM 6 and
the standard (Supplementary Fig. 9). Supplementation with 381 ± 30 μM 2, with a ratio of around 1:3.6:9.8, were produced
2,4-dinitrophenyl hydrazine (DNP) for derivatization led to identi- by 20 μM NosL catalysis over a 2-hour period (Supplementary
fication of the second shunt product as glyoxylate (7) (correspond- Fig. 4b). For conversion of the substrate L-tryptophan under these
ing to 8, the derivative 2-(2,4-dinitrophenyl) hydrazonoacetate, conditions, one molecule of NosL produced approximately two
Fig. 2c and Supplementary Fig. 10). Thus, the first Cα-Cβ cleav- molecules of 1 along with seven molecules of 6. The production of
age of L-tryptophan appears to provide the 3-methylindole part, AdoH was consistently higher, showing a yield ratio around 2.1:1
whereas the readdition-coupled, second cleavage may take place for 2, corresponding to a sum of 1 and 6. For optimizing the reac-
on the separated 2C-N unit to furnish the 2-carboxylate group. tion, we speculated that the degree of reduction in the reaction
Consistent with this prediction, upon a similar derivatization, we mixture may have an impact on the proportion of 1. We therefore
detected the production of formaldehyde (9) (corresponding to 10, measured the effect of the concentration of the chemical reduc-
the derivative 1-(2,4-dinitrophenyl)-2-methylene hydrazine, Fig. 2c tant dithionite on the yields of 1 and 6 (Supplementary Fig. 4c).
and Supplementary Fig. 11), which most likely comes from the Intriguingly, 6 production was highly dependent on dithionite con-
α-carbon of L-tryptophan.
centration, whereas the yields of 1 were relative constant, around
156
nature chemical biology | vol 7 | march 2011 | www.nature.com/naturechemicalbiology

NATURE ChEMICAL bIOLOgy doi: 10.1038/nchembio.512
article
NH2
To experimentally support the hypothesis, we attempted to probe
the putative intermediate in the NosL-catalyzed reaction by using
a derivatization-associated, rapid-quench method previously used
for analyzing the glutamate mutase–catalyzed reaction. Glutamate
mutase, an Ado•-producing adenosylcobalamin (AdoCbl, coen-
zyme B12)-dependent protein, has been demonstrated to convert
L-glutamic acid into glycyl radical (trapped as glycine by using this
quench technique) and acrylate during L-threo-3-methylaspartate
formation18–20. The NosL-catalyzed reaction was quenched at 10 sec
before derivatization with dansyl chloride. HPLC-MS analysis
showed the production of glycine (21) (corresponding to the deri-
vative glycine-dansyl, 22, Fig. 2d and Supplementary Fig. 14),
as well as methionine (23) (corresponding to the derivative
methionine-dansyl, 24, Fig. 2d and Supplementary Fig. 15) deriv-
ing from S-AdoMet. Using asparagine as an internal standard, the
glycine species was observed in a yield of 2 ± 0.5 μM, equal to around
2.5% of the enzyme active site. Under highly reductive conditions,
this species is presumed to derive from the immediately uncoupled
intermediate, glycyl radical, consistent with the predicted manner
of homolytic cleavage of the Cα-Cβ bond of L-tryptophan during
1 formation.
COOH
COOH
N
H
N
H
L-[1-13C]-Tryptophan
3
NH2
COOH
COOH
N
H
N
H
NosL
(MIA synthase)
L-[1-13C]-Tryptophan
4
H2N
D
D
D
H
D
D
D
D
COOH
D
D
D
D
D
D
COOH
N
N
H
H
Substrate flexibility of NosL
D
The characterization of the NosL chemistry then prompted us to
feed available analogs of L-tryptophan, including D-tryptophan
and derivatives with different substitutions on the indole ring, into
SL4101 to determine the substrate tolerance of NosL. Individual
additions of most of these substrates had no effect on 1 production
and failed to afford the expected 1 analogs (Supplementary Table 3).
These indicate the stringent substrate specificity of NosL in stereo-
chemistry and in functionalization of the indole ring. In contrast,
feeding of 5- or 6-fluoro-DL-tryptophan (only the L-configuration
isomer can be used as the substrate) led to generation of the distinct
product with a yield approximately 20% of the concomitant 1 pro-
duction (Supplementary Fig. 16). Spectroscopic analyses, includ-
ing HR-ESI-MS and 1H, 13C and 19F NMR (Supplementary Fig. 17),
clearly established these compounds as corresponding to 5-fluoro-
MIA (25) and 6-fluoro-MIA (26), respectively (Scheme 1).
L-[2H8]-Tryptophan
5
NH2
COOH
COOH
F
F
N
H
N
H
5 or 6-fluoro-L-Tryptophan
25 or 26
Scheme 1 | NosL-catalyzed carbon chain reconstitution of L-tryptophan
shown by labeling patterns and generation of fluorinated MIA analogs.
The solid rectangle shows the 13c-labeled carbon atom. D, deuterium.
F, fluorine.
30–42 μM in all assays. In particular, when 100 μM dithionite was generation of a fluorinated NOS analog
used, the ratio of 1 to 6 can be improved to around 3:1.
To determine whether or not the fluorinated MIA (1) can be incor-
porated as an intermediate in vivo, we fed 5-fluoro-DL-tryptophan
into the NOS-producing strain S. actuosus. HPLC-MS analysis
Prediction of the Ca-Cb bond cleavage manner
Inspired by identification of 3-methylindole (6) and glyoxylate (7) showed a dramatic decrease in NOS production (20–25% relative
as the shunt products, we proposed that the Ado•-initiated carbon
chain reconstitution of L-tryptophan may begin with a Cα-Cβ
Heterolytic cleavage
Homolytic cleavage
bond cleavage during NosL-catalyzed 1 formation. Depending on
the bond-breaking pattern—that is, whether it is via homolysis or
via heterolysis—there are two sets of hypothetical intermediates
that share a common fate in generating the shunt product pair
(Fig. 3). Thus, the density functional theory (DFT) calculation
was performed to differentiate between these two possibilities
(Fig. 3 and Supplementary Figs. 12 and 13). Although the direct
cleavage of the L-tryptophan neutral radical (11) as outlined in
Path 1 leads to the generation of the intermediates 12 and 13
(heterolytic) or 14 and 15 (homolytic), Path 2 indicates that the
cleavage accompanied by an intramolecular hydrogen migration
results in the production of the intermediates 16 and 17 (hetero-
lytic) or 18 and 19 (homolytic). Given that the intermediate 11
may get a proton from the environment, the model shown in
Path 3 is also considered, indicating the generation of the inter-
mediates 16 and 20 (heterolytic) or 18 and 15 (homolytic). At
all events the homolytic products are lower in energy than their
corresponding heterolytic products, suggesting that the homo-
lysis was thermodynamically favored to form 3-methyleneindole
and glycyl radical.
OH
H2
N
OH
O
H2
N
Direct cleavage
Path 1
+
+
O
N
N
12
13
14
15
68.6
–0.8
NH2
COOH
Cleavage with an
intramolecular
H-migration
HN
OH
O
OH
O
HN
+
+
N
H
N
H
N
Path 2
16
17
18
19
11
0.0
–34.6
Most favorable intermediates
Obtaining a proton
from the environment
H2N
OH
O
+ H+
H2
N
OH
O
+
+
N
N
H
Path 3
H
16
20
18
15
0.0
–6.5
Figure 3 | Different patterns for cleaving the Ca-Cb bond of L-tryptophan
neutral radical 11. The DFT calculation–based relative free energies
(including solvent effect ΔGsol) are in kcal per mol. In Path 1 and Path 2,
the energy of 16 + 17 is used as the reference zero energy, whereas
in Path 3 the energy of 16 + 20 is used as the reference.
nature chemical biology | vol 7 | march 2011 | www.nature.com/naturechemicalbiology
157

NATURE ChEMICAL bIOLOgy doi: 10.1038/nchembio.512
article
DISCUSSION
I
NOS
The radical S-AdoMet superfamily currently comprises thousands
of proteins that participate in numerous biochemical processes in
animals, plants and microorganisms17,21–24. These proteins share a
common mechanism to generate a powerful oxidant, Ado•, thereby
initiating highly diverse transformations relevant to DNA repair
and in the biosynthesis of vitamins, coenzymes and antibiotics.
Biochemical mechanisms for most of such conversions have not
been characterized, owing to a lack of information about the enzyme
functions and radical-mediated transformations. Focusing on the
characterization of NosL and NosL-catalyzed reactions, we herein
unveiled a radical-mediated, unusual fragmentation-recombination
process, which represents a key step in understanding radical
S-AdoMet enzyme-catalyzed complex structure rearrangements.
We propose a mechanism for NosL-catalyzed reaction, as
described below. Distinct from the widely found C-H bond cleavage
in the reactions controlled by known radical S-AdoMet enzymes17,
II
5′-fluoro-NOS (27)
0
5
10
15
20
25
30
Time (min)
Figure 4 | Production of NOS and fluorinated thiopeptide. hPlc analysis
of the fermentation cultures of S. actuosus without supplementation (I)
and with supplementation with 5-fluoro-Dl-tryptophan (II). all the
examinations were performed in duplicate (each had two parallel samples).
to that of the wild-type strain, Fig. 4), presumably because of com- Ado• in NosL catalysis may abstract the hydrogen atom from N1
petitive inhibition of biosynthetic enzymes in the substrates. A new on the indole ring of L-tryptophan to generate 11, the neutral sub-
product, with UV spectra similar to NOS, was clearly observed, strate radical. The homolysis is thermodynamically favorable, lead-
showing a yield approximately 20% of the concomitant NOS pro- ing to the Cα-Cβ bond cleavage for the first fragmentation, which
duction. According to the molecular formula, C51H42FN13O12 gives the transient units 3-methyleneindole and glycine radical.
(HR-ESI-MS m/z calculated = 1,238.13114, found 1,238.12959), Taking a simultaneous proton transfer into account, the resulting
this compound was deduced to be a NOS analog substituted by a cationic 3-methyleneindole (18) and glycine radical (19) are more
fluorine atom at C5′ of the indole ring (Fig. 1a and Supplementary stable, facilitating their subsequent readdition to generate the radi-
Fig. 18). For structural elucidation, it was purified and then sub- cal intermediate 28 (Scheme 2, Mechanism 1). Elimination of the
jected to comparative spectroscopic analysis with NOS, showing Cα-N unit (giving the methanimine radical 29) from 28 can pro-
that the only difference was in the indole moiety. 19F NMR and 1D ceed to furnish the 2-carboxylate group to yield 1, and the hydro-
and 2D NMR spectra further supported this structure assignment gen rebound to 29 results in the production of CH2=NH, which is
of 5′-fluoro-NOS (27, Supplementary Fig. 19 and Supplementary hydrolyzed in the aqueous medium to form formaldehyde (9) and
Table 4). The effect of the 5′-fluoro-substitution on antibacterial ammonia. Alternatively, according to a glycine decarboxylation–
activity was consequently evaluated by bioassays against the test based free-radical mechanism proposed previously25, the second
strain Bacillus subtilis, showing the minimum inhibitory concen- fragmentation on the separated 2C-N unit (glycyl radical 15)
tration at 0.004 μM ml−1 for 27 to that at 0.008 μM ml−1 for NOS could precede the readdition, resulting in a direct reposition of
(Supplementary Table 5). Notably, there has yet to be a halogenated the carboxylate at C2 of the indole ring (Scheme 2, Mechanism 2):
member of the naturally occurring thiopeptide antibiotics, which (i) the protonated radical 15 may undergo a C-C bond cleav-
number over 80 compounds reported to date1.
age to generate carboxyl radical 30; and (ii) the addition of 30 to
–
HN
–
HN
NH
OH
O
OH
O
+
COOH
OH
N
N
H
N
O
+
H
H
19
HC NH
18
28
29
Mechanism 1
H2C NH
NH2
COOH
NH2
COOH
O
HCHO
9
+
COOH
+
COOH
N
H
N
H
7
6
NH3
N
N
1
H
11
Ado
AdoH
[Fe4S4]2+
L-Methionine
Mechanism 2
+
COOH
COOH
[Fe4S4]+
H2N
COOH
N
N
N
H
15
S-AdoMet
31
14
H+
+
CO2H
30
H2N
COOH
HCHO
+
9
+
H2C NH2
NH3
Scheme 2 | Mechanistic hypothesis for NosL-catalyzed conversion. For mechanism 1, the recombination may take place between cationic 18 and
radical 19 followed by elimination of the cα-N unit from the resulting radical 28, and for mechanism 2, the first separated radical 15 could undergo the
second fragmentation to generate carboxyl radical 30 before the addition onto 14.
158
nature chemical biology | vol 7 | march 2011 | www.nature.com/naturechemicalbiology

NATURE ChEMICAL bIOLOgy doi: 10.1038/nchembio.512
article
Time course–based analysis of MIA production in vivo. The harvested SL4101
cells described above were resuspended in a nutrient-insufficient medium
(yeast extract 0.1% (w/v), tryptone 0.5% (w/v), glucose 0.4% (w/v), NaCl 0.5%
(w/v), glutamine 0.05% (w/v), K2HPO4 0.2% (w/v), MgSO4 0.01% (w/v) and
FeSO4 0.001% (w/v)), with OD600 nm of 0.6–0.8. Variable amino acids, including
L-tryptophan, L-serine and L-glycine, were individually added to the broth (5 mM
in final) before further incubation at 28 °C. MIA production was measured by
HPLC at 0.5, 1, 2, 3, 4, 10 or 20 h. The MIA production is approximately linear
with respect to time in initial 4 h, and the apparent rate was accordingly estimated.
3-methyleneindole 14 can yield radical 31, which, when hydrogen
is rebound, eventually leads to the production of 1.
NosL-catalyzed in vitro conversion produced 1 along with two
shunt products, 3-methylindole (6) and glyoxylate (7), which may
originate from the intermediate pair 3-methyleneindole and glycyl
radical, respectively. We proposed that the biotransformation after
a Cα-Cβ bond cleavage, such as the inefficient recombination, may
serve as a limitation step in NosL-catalyzed in vitro process. The
overproduced intermediates could be released from the active site
of NosL into the aqueous medium: while 3-methyleneindole is aro-
matized by reduction to 6, glycyl radical is rapidly degraded to 7
and ammonia. Organic radicals with one unpaired electron have
been known to be highly active and unstable. For instance, if the
putative substrate-based glycyl radical (Scheme 2) fails to be further
processed (to be added onto the intermediate 3-methyleneindole,
as proposed), it may lose an electron to generate dehydroglycine
and then undergo hydrolysis for glyoxylate production. A similar
result has been found in the in vitro reaction catalyzed by ThiH26.
Without the associated components for turning over glycyl radical
Feeding of isotope-labeled L-tryptophans and variable L-tryptophan analogues.
The procedure is similar to that of the time course–based analysis for MIA
production, except the individual supplementation of isotope-labeled substrates
(that is, L-[2H8]-tryptophan, L-[1-13C]-tryptophan and L-[3-13C]-tryptophan),
D-tryptophan and derivatives with different substitution on the indole ring
(that is, 1-methyl-L-tryptophan, 2-methyl-DL-tryptophan, 4-methyl-DL-tryptophan,
6-methyl-DL-tryptophan, 5-hydroxyl-L-tryptophan, 5-methoxy-DL-tryptophan,
5-bromo-DL-tryptophan, 5-fluoro-DL-tryptophan and 6-fluoro-DL-tryptophan)
to a concentration of 1 mM before further incubation at 28 °C for 8 h. The
production of labeled or functionally substituted MIAs was monitored by
HPLC-MS.
Structural characterization of MIA, its analogs and a new NOS analog by
feeding 5-fluoro-DL-tryptophan into the NOS-producing strain is summarized in
or dehydroglycine in thiazole formation, ThiH catalyzes the Cα-Cβ Supplementary Methods.
bond cleavage of L-tyrosine and gives p-cresol and glyoxylate as
the products.
Reconstitution of NosL. Production and anaerobic purification of NosL are
summarized in Supplementary Methods. A previously reported procedure31
was modified to reconstitute the active enzyme. Dithiothreitol was added to the
purified protein (10 mM final). Under this reductive condition, Fe(NH4)2(SO4)2
solution (50 mM) was added carefully into the suspension to a final concentration
of 500 μM. After 10 min, Na2S solution (50 mM) was added in the same way to
a concentration of 500 μM. The solution had been incubated on ice for 5–7 h.
The resulting dark brown suspension was then subjected to desalting on a column
(10-DG, Bio-Rad) that was pre-equilibrated with the elution buffer (50 mM
Tris-HCl, 25 mM NaCl, 10 mM dithiothreitol and 10% (v/v) glycerol, pH 8.0).
The colored fraction was collected and concentrated to 100 μM for in vitro assay.
Protein characterization is described in Supplementary Methods.
Substrate fragmentation is known in Ado•-induced biotransfor-
mations, as exemplified by ThiH and by newly characterized HydG
(24% identity to NosL)27, which catalyzes the L-tyrosine cleavage to
generate p-cresol and cyanide. NosL, ThiH and HydG may share
a common paradigm to process the radical-based, aromatic amino
acid substrates for the Cα-Cβ bond cleavage, despite the differ-
ence in the fate of the resulting glycyl radical (or dehydroglycine)
intermediate. However, the fragmentation-recombination found in
NosL chemistry is rare. To our knowledge, the only known example
is glutamate mutase, the radical AdoCbl–dependent protein that
converts L-glutamic acid to the β-methyl branched product (but
without fragment elimination)20,24. This strategy might be used in
certain Ado•-mediated structural rearrangements, such as the ThiC-
catalyzed complex conversion of 5-aminoimidazole ribonucleotide
to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate in
thiamine biosynthesis28.
Assay of the NosL activity in vitro. The 100 μl of reaction mixture contained
10 mM dithiothreitol, 500 μM dithionite, 200 μM S-AdoMet, 200 μM L-tryptophan
(or L-[2H8]-tryptophan) and 20 μM reconstituted NosL in 50 mM Tris-HCl buffer
(pH 8.0). Reactions were initiated by adding S-AdoMet to the 10-min preincubated
mixture (as a negative control that contained the components except S-AdoMet);
the mixture was then incubated at 25 °C for 2 h. To terminate the reaction, trifluor-
oacetic acid (TFA) was added to a final concentration of 10% (v/v) to inactivate the
enzyme. After removal of the precipitates by centrifugation, the supernatant was
subjected to HPLC-MS analysis. For evaluation of a natural reduction system, the
chemical reductant dithionite was replaced by 0.5 mM NADPH, 50 μM flavodoxin
and 20 μM flavodoxin reductase.
In conclusion, we have uncovered NosL as a MIA synthase
that catalyzes an unprecedented carbon side chain reconstitution
of L-tryptophan. The radical-mediated, unusual fragmentation-
recombination process may be general for radical S-AdoMet pro-
tein-catalyzed structural rearrangements in certain uncharacterized
biotransformations. Taking advantage of the substrate tolerance
of NosL, regiospecific fluorination of NOS could be achieved via
a modified MIA intermediate. The NOS analog produced in this
fashion and bearing fluorine at the 5′ position showed improved
antibacterial activity. This application of combinatorial biosynthe-
sis, enabled by our elucidation of NosL chemistry, complements
recent advances in understanding sequence permutations of the
For GC-MS analysis, the supernatant was further extracted by ethyl acetate.
For derivatization, DNP was added to the supernatant (10 mM final), which was
further incubated at 37 °C for 30 min.
Quantitative analysis of product formation in vitro. The 100 μl of reaction mixture
contained 10 mM dithiothreitol, 1 mM dithionite, 1 mM S-AdoMet and 500 μM
L-tryptophan, in 50 mM Tris-HCl buffer (pH 8.0). The reconstituted NosL, varying
in concentration (10 μM, 20 μM, 40 μM or 80 μM), was added into each reaction
mixture. The 20 μM NosL catalysis was used for a time-course analysis (by
terminating at 10 min, 30 min, 60 min, 90 min or 120 min). To measure the effect
of dithionite concentration on the yields of MIA and 3-methylindole, each 100 μM
of reaction mixture contained 20 μM reconstituted NosL, 10 mM dithiothreitol,
1 mM SAM, 500 μM L-tryptophan and dithionite varying in concentration
(100 μM, 200 μM, 500 μM, 1 mM, 2 mM or 4 mM) in 50 mM Tris-HCl buffer
(pH 8.0). The workups for the initiation, incubation and termination of reactions
and for the analysis of products were identical to those described above.
precursor peptide29,30
.
METhODS
Bacterial strains, plasmids and primers. Please see Supplementary Tables 1 and 2.
Materials. Please see Supplementary Methods.
Probing of the putative intermediate in NosL-catalyzed conversion. Trapping of
glycine species in NosL-catalyzed conversion was performed according to a modi-
fied procedure18. The 100 μl of reaction mixture contained 20 mM dithiothreitol,
2 mM dithionite, 1 mM S-AdoMet, 1 mM L-tryptophan and 80 μM reduced NosL
in 50 mM Tris-HCl buffer (pH 8.0). Reactions were initiated by adding S-AdoMet
to the 10-min preincubated mixture (as a negative control containing the compo-
nents except S-AdoMet); the mixture was mixed immediately and then terminated
at 10 sec by addition of TFA to a final concentration of 15% (v/v) to precipitate the
enzyme. After removal of the precipitate by centrifugation, Na2CO3 (1.5 M final)
and dansyl chloride (1 mM final) were added to the supernatant, which was further
incubated at 50 °C for 60 min. We then added 20 μl of TFA to the solution, and the
dansyl derivatives were subjected to HPLC-MS analysis. For quantifying the yield
of glycine species, asparagine (20 μM) was used as an internal standard.
Production of MIA in vivo. Construction of the recombinant strain SL4101 for
expressing nosL is described in Supplementary Methods. SL4101 was cultured
in LB medium. Once the cell optical density (OD) reached 0.6–0.8 at 600 nm, the
Fe(NH4)2(SO4)2 solution was added into the culture broth to a concentration of
50 μM, and then the cells were incubated on ice for 10 min. After addition of
isopropyl-β-D-thiogalactopyranoside (100 μM in final), nosL expression had been
induced at 25 °C for 6–8 h. After the cells were harvested by centrifugation for the
following experiments (see below), the supernatant was directly subjected to HPLC
analysis for product examination. SL4100 carrying the vector pET28a alone was used
as the control in the parallel analytic process. Site-specific mutagenesis for exploring
the cofactor-binding nature of NosL is described in Supplementary Methods.
nature chemical biology | vol 7 | march 2011 | www.nature.com/naturechemicalbiology
159

NATURE ChEMICAL bIOLOgy doi: 10.1038/nchembio.512
article
biosynthetic pathways with radical mechanisms: functional characterization
using new analysis and information visualization methods. Nucleic Acids Res.
29, 1097–1106 (2001).
received 17 May 2010; accepted 1 December 2010;
published online 16 January 2011
22. Nicolet, Y. & Drennan, C.L. AdoMet radical proteins–from structure to
evolution–alignment of divergent protein sequences reveals strong
secondary structure element conservation. Nucleic Acids Res. 32, 4015–4025
(2004).
references
1. Bagley, M.C., Dale, J.W., Merritt, E.A. & Xiong, X. iopeptide antibiotics.
Chem. Rev. 105, 685–714 (2005).
2. Arndt, H.-D., Schoof, S. & Lu, J.-Y. iopeptide antibiotic biosynthesis.
Angew. Chem. Int. Ed. Engl. 48, 6770–6773 (2009).
23. Wang, S.C. & Frey, P.A. S-adenosylmethionine as an oxidant: the radical SAM
superfamily. Trends Biochem. Sci. 32, 101–110 (2007).
3. Li, C. & Kelly, W.L. Recent advances in thiopeptide antibiotic biosynthesis.
Nat. Prod. Rep. 27, 153–164 (2010).
24. Marsh, E.N.G., Patterson, D.P. & Li, L. Adenosyl radical: reagent and catalyst
in enzyme reactions. ChemBioChem 11, 604–621 (2010).
25. BonifaČić, M., Stefanic, I., Hug, G.I., Armstrong, D.A. & Asmus, K.-D.
Glycine decarboxylation: the free radical mechanism. J. Am. Chem. Soc. 120,
9930–9940 (1998).
4. Nicolaou, K.C., Chen, J.S., Edmonds, D.J. & Estrada, A.A. Recent advances in
the chemistry and biology of naturally occurring antibiotics. Angew. Chem.
Int. Ed. Engl. 48, 660–719 (2009).
5. Wieland Brown, L.C., Acker, M.G., Clardy, J., Walsh, C.T. & Fischbach, M.A.
irteen posttranslational modifications convert a 14-residue peptide into the
antibiotic thiocillin. Proc. Natl. Acad. Sci. USA 106, 2549–2553 (2009).
6. Liao, R. et al. iopeptide biosynthesis featuring ribosomally synthesized
precursor peptides and conserved posttranslational modifications. Chem. Biol.
16, 141–147 (2009).
26. Kriek, M., Martins, F., Challand, M.R., Crof, A. & Roach, P.L. iamine
biosynthesis in Escherichia coli: identification of the intermediate and
by-product derived from tyrosine. Angew. Chem. Int. Ed. Engl. 46, 9223–9226
(2007).
27. Driesener, R.C. et al. [FeFe]-hydrogenase cyanide ligands derived from
S-adenosylmethionine-dependent cleavage of tyrosine. Angew. Chem. Int. Ed.
Engl. 49, 1687–1690 (2010).
7. Kelly, W.L., Pan, L. & Li, C. iostrepton biosynthesis: prototype for a new
family of bacteriocins. J. Am. Chem. Soc. 131, 4327–4334 (2009).
8. Morris, R.P. et al. Ribosomally synthesized thiopeptide antibiotics targeting
elongation factor Tu. J. Am. Chem. Soc. 131, 5946–5955 (2009).
9. Yu, Y. et al. Nosiheptide biosynthesis featuring a unique indole side ring
formation on the characteristic thiopeptide framework. ACS Chem. Biol. 4,
855–864 (2009).
10. Wang, J. et al. Identification and analysis of the biosynthetic gene cluster
encoding the thiopeptide antibiotic cyclothiazomycin in Streptomyces
hygroscopicus 10–22. Appl. Environ. Microbiol. 76, 2335–2344 (2010).
11. Ding, Y. et al. Moving posttranslational modifications forward to
biosynthesize the glycosylated thiopeptide nocathiacin I in Nocardia sp.
ATCC202099. Mol. Biosyst. 6, 1180–1185 (2010).
12. Houck, D.R., Chen, L.-C., Keller, P.J., Beale, J.M. & Floss, H.G. Biosynthesis
of the modified peptide antibiotic nosiheptide in Streptomyces actuosus.
J. Am. Chem. Soc. 110, 5800–5806 (1988).
13. Mocek, U. et al. Biosynthesis of the modified peptide antibiotic nosiheptide
in Streptomyces actuosus. J. Am. Chem. Soc. 115, 7557–7568 (1993).
14. Mocek, U. et al. Biosynthesis of the modified peptide antibiotic thiostrepton
in Streptomyces azureus and Streptomyces laurentii. J. Am. Chem. Soc. 115,
7992–8001 (1993).
15. Smith, T.M., Priestley, N.D., Knaggs, A.R., Nguyen, T. & Floss, H.G.
3,4-Dimethylindole-2-carboxylate and 4-(1-Hydroxyethyl)-2-
quinolinecarboxylate activating enzymes from the nosiheptide and
thiostrepton producers, Streptomyces actuosus and Streptomyces laurentii.
J. Chem. Soc. Chem. Commun. 21, 1612–1614 (1993).
28. Chatterjee, A. et al. Reconstitution of iC in thiamine pyrimidine
biosynthesis expands the radical SAM superfamily. Nat. Chem. Biol. 4,
758–765 (2008).
29. Acker, M.G., Bowers, A.A. & Walsh, C.T. Generation of thiocillin variants by
prepeptide gene replacement and in vivo processing by Bacillus cereus. J. Am.
Chem. Soc. 131, 17563–17565 (2009).
30. Bowers, A.A., Acker, M.G., Koglin, A. & Walsh, C.T. iazolyl peptide
antibiotic biosynthesis: a cascade of posttranslational modifications on
ribosomal nascent proteins. J. Am. Chem. Soc. 132, 7519–7527 (2010).
31. Kriek, M. et al. iazole synthase from Escherichia coli - an investigation of
the substrates and purified proteins required for activity in vitro. J. Biol.
Chem. 282, 17413–17423 (2007).
acknowledgments
We thank H.G. Floss (University of Washington) for providing S. actuosus ATCC25421
and for his pioneering work on NOS biosynthesis and Y. Zhang and W. Tong, (High
Magnetic Field Laboratory, Chinese Academy of Sciences) for assistance with EPR
analysis. This work was supported in part by grants from US National Institutes of Health
(CA094426 to B.S.), Chinese National Natural Science Foundation (20832009, 30525001,
90713012 and 20921091), Chinese Ministry of Science and Technology (2009ZX09501-
008), Chinese National Basic Research Program (“973 program,” 2010CB833200),
Chinese Academy of Sciences (KJCX2-YW-H08 and KSCX2-YW-G-06) and Science and
Technology Commission of Shanghai Municipality (09QH1402700) of China (all to W.L).
16. Priestley, N.D. et al. Studies on the biosynthesis of thiostrepton:
4-(1-hydroxyethyl)-quinoline-2-carboxylate as a free intermediate on the
pathway to the quinaldic acid moiety. Bioorg. Med. Chem. 4, 1135–1147 (1996).
17. Frey, P.A., Hegeman, A.D. & Ruzika, F.J. e radical SAM superfamily.
Crit. Rev. Biochem. Mol. Biol. 43, 63–88 (2008).
author contributions
Q.Z., D.C., Y.Y. and L.D. carried out the experiments; Y.L. performed the theoretical
calculations; Q.Z., B.S. and W.L. analyzed the data and wrote the paper; and W.L.
designed the research. All authors discussed results and approved the final manuscript.
18. Chih, H.W. & Marsh, E.N.G. Pre-steady-state kinetic investigation of
intermediates in the reaction catalyzed by adenosylcobalamin-dependent
glutamate mutase. Biochemistry 38, 13684–13691 (1999).
competing financial interests
19. Chih, H.-W. & March, N.G. Mechanism of glutamate mutase: Identification
and kinetic competence of acrylate and glycyl radical as intermediates in the
rearrangment of glutamate to methylaspartate. J. Am. Chem. Soc. 122,
10732–10733 (2000).
20. Banerjee, R. Radical carbon skeleton rearrangements: catalysis by coenzyme
B12-dependent mutases. Chem. Rev. 103, 2083–2094 (2003).
21. Sofia, H.J., Chen, G., Hetzler, B.G., Reyes-Spindola, J.F. & Miller, N.E. Radical
SAM, a novel protein superfamily linking unresolved steps in familiar
The authors declare no competing financial interests.
additional information
Supplementary information and chemical compound information is available online at
http://www.nature.com/naturechemicalbiology/. Reprints and permissions information
is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence
and requests for materials should be addressed to W.L.
160
nature chemical biology | vol 7 | march 2011 | www.nature.com/naturechemicalbiology