The Catalytic Mechanism of the Class C Radical S-Adenosylmethionine Methyltransferase NosN

Article abstract of DOI:10.1002/anie.201609948

S-Adenosylmethionine (SAM) is one of the most common co-substrates in enzyme-catalyzed methylation reactions. Most SAM-dependent reactions proceed through an S_N2 mechanism, whereas a subset of them involves radical intermediates for methylating non-nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM-dependent methyltransferases, NosN does not produce S-adenosylhomocysteine (SAH) as a co-product. Instead, NosN converts SAM into 5′-methylthioadenosine as a direct methyl donor, employing a radical-based mechanism for methylation and releasing 5′-thioadenosine as a co-product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme-catalyzed methylation reactions.

Full text of DOI:10.1002/anie.201609948

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International Edition: DOI: 10.1002/anie.201609948
German Edition: DOI: 10.1002/ange.201609948
Biosynthesis Very Important Paper
The Catalytic Mechanism of the Class C radical S-Adenosylmethionine
Methyltransferase NosN
Wei Ding, Yongzhen Li, Junfeng Zhao, Xinjian Ji, Tianlu Mo, Haocheng Qianzhu, Tao Tu,
Zixin Deng, Yi Yu, Fener Chen, and Qi Zhang*
Abstract: S-Adenosylmethionine (SAM) is one of the most
common co-substrates in enzyme-catalyzed methylation reac-
tions. Most SAM-dependent reactions proceed through an SN2
mechanism, whereas a subset of them involves radical
intermediates for methylating non-nucleophilic substrates.
Herein, we report the characterization and mechanistic inves-
tigation of NosN, a class C radical SAM methyltransferase
involved in the biosynthesis of the thiopeptide antibiotic
nosiheptide. We show that, in contrast to all known SAM-
dependent methyltransferases, NosN does not produce S-
of various non-nucleophilic substrates.[2] While most radical
SAM methyltransferases (RSMTs) share a similar strategy in
using SAM both as a methyl donor and a radical initiator, the
catalytic mechanisms of RSMTs are diverse.[2]
NosN is a radical SAM enzyme that is involved in the
biosynthesis of nosiheptide (1),[4] a clinically interesting
thiopeptide antibiotic produced by Streptomyces actuosus
(Figure 1A).[5] Nosiheptide belongs to the large class of
adenosylhomocysteine (SAH) as
a co-product. Instead,
NosN converts SAM into 5’-methylthioadenosine as a direct
methyl donor, employing a radical-based mechanism for
methylation and releasing 5’-thioadenosine as a co-product.
A series of biochemical and computational studies allowed us
to propose a comprehensive mechanism for NosN catalysis,
which represents a new paradigm for enzyme-catalyzed
methylation reactions.
M
ethyltransferases are prevalent in biology and are
essential for almost all life processes. The vast majority of
methyltransferases use S-adenosylmethionine (SAM) as the
methyl donor, and typically the methyl group is transferred
from SAM to a nucleophile through an SN2 mechanism.[1]
Methylation can also occur at non-nucleophilic centers such
as inert carbon or phosphorous atoms through radical-based
mechanisms.[2] Currently, all of the known radical-based
methyltransferases belong to the radical SAM superfamily,
a large enzyme superfamily consisting of more than 16,500
members found in all three domains of life.[3] Radical SAM
enzymes utilize a [4Fe-4S] cluster to bind SAM and reduc-
tively cleave its carbon–sulfur bond to produce a highly
reactive 5’-deoxyadenosyl (dAdo) radical, which initiates
a highly diverse array of reactions, including the methylation
Figure 1. Functional diversity of the class C RSMTs. A) The biosynthe-
sis of nosiheptide in S. actuosus involves the class C RSMT NosN. The
carbon atom introduced by NosN is indicated with a solid triangle.
The nosN-knockout mutant of S. actuosus does not produce nosihep-
tide, but rather the nosiheptide analogue 2 (Figure S1–S3). B) Bayesian
MCMC phylogeny of the class C RSMTs and other HemN-like enzymes
(for details of the phylogenetic analysis, see Methods in the Support-
ing Information).
[*] Prof. Dr. W. Ding, Dr. Y. Li, J. Zhao, X. Ji, T. Mo, H. Qianzhu,
Prof. Dr. T. Tu, Prof. Dr. F. Chen, Prof. Dr. Q. Zhang
Department of Chemistry, Fudan University
Shanghai, 200433 (China)
E-mail: qizhang@sioc.ac.cn
Prof. Dr. W. Ding
School of Life Sciences, Lanzhou University
Lanzhou, 730000 (China)
ribosomally synthesized and posttranslationally modified
peptides (RiPPs).[6] It contains a precursor-peptide-derived
macrocyclic ring that consists of a central tetrasubstituted
pyridine and five thiazoles, and a ribosomally-independent
side ring system containing an indolic acid moiety (Fig-
ure 1A). Previous study have shown that the nosN-knockout
mutant of S. actuosus does not produce nosiheptide but rather
the nosiheptide analogue 2, in which the indolic side ring
system is incomplete (Figure 1A), thus suggesting that NosN
J. Zhao, Prof. Dr. Z. Deng, Prof. Dr. Y. Yu
Key Laboratory of Combinatory Biosynthesis and Drug Discovery
(Ministry of Education), School of Pharmaceutical Sciences
Wuhan University, Wuhan, 430071 (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201609948.
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is likely responsible for appending a methyl group on the C4
of the indolyl moiety during nosiheptide maturation.[4]
The fact that NosN is a methyltransferase is further
supported by the observation that this enzyme shares
significant sequence similarity with other known methyltrans-
ferases, such as PbtM2 and PbtM3, which are involved in
GE2270 biosynthesis,[7] and YtkT, which is involved in
yatakemycin biosynthesis.[8] NosN, YtkT, PbtM2, PbtM3,
and some other putative methyltransferases are homologous
to the coproporphyrinogen III oxidase HemN (Figure 1B),
and are classified as class C RSMTs.[2a,c] Notably, HemN
catalyzes two sequential steps of oxidative decarboxylation of
coproporphyrinogen III,[9] a reaction that is totally unrelated
to methylation. Although the methyltransferase activity of
YtkT has been successfully reconstituted in vitro by Tang and
co-workers,[8] and ChuW has very recently been reconstituted
by Lanzilotta and co-workers,[10] the catalytic mechanisms of
the class C RSMTs remain largely unknown.
To interrogate the function and mechanism of NosN, the
nosN gene was amplified from the genomic DNA of S.
actuosus and expressed in Escherichia coli with an N-terminal
hexa-histidine tag, and the protein was purified by Ni2+
affinity chromatography under strictly anaerobic conditions.
After chemical reconstitution of the [4Fe-4S] cluster, followed
by gel-filtration, the protein was found to contain 4.5 Æ
0.2 molFe and 4.8 Æ 0.3 molS per mol protein. UV/Vis
spectroscopy showed that the protein solution had a broad
absorption around 415 nm (Figure S4), a feature character-
istic of [4Fe-4S]-containing proteins. Analysis of the reaction
mixture containing SAM, sodium dithionite, and the recon-
stituted NosN showed that 5’-deoxyadenosine (dAdoH) was
produced in the assay mixture (Figure 2A, trace ii), thus
suggesting that NosN is a radical SAM enzyme.
Figure 2. Mechanistic investigation of NosN. A) HPLC analysis of the
NosN assay mixture, using a supernatant with boiled NosN as
a negative control (i), and reconstituted NosN with the other required
components in the absence (ii) or presence (iii) of 4. Because of the
low yield and the similar retention time to that of dAdoH, tAdoH is
not visible in this analysis. B) Structures of the indolyl compounds in
relation to our biochemical analysis. C) MS spectra of 5 (i) and 5d (ii)
produced in the NosN-catalyzed reaction with SAM or d3-SAM,
respectively. D) Chemical structures of the MTA and tAdoH produced
in the NosN reaction. E) LC–MS analysis of NosN reaction mixtures,
showing the extracted ion chromatograms (EICs) of [M+H]+ =284.1
(corresponding to tAdoH) for the control reaction with boiled NosN
(i), the NosN reaction (ii), and a synthetic tAdoH standard (iii).
A possible substrate of NosN is 3-methyl-2-indolic acid
(MIA, 3), a nosiheptide biosynthetic intermediate produced
from l-tryptophan by the radical SAM enzyme NosL (Fig-
ure 1A).[11] To test whether MIA is the NosN substrate, we
performed the NosN assays with MIA and carefully examined
the assay mixture by liquid chromatography high resolution
mass spectrometry (LC–HRMS). No methylated product was
found in the assay mixture, thus suggesting that MIA is
probably not the substrate of NosN. Another possible
substrate is 2, which is produced by the nosN-knockout
mutant (Figure 1A). We obtained the purified compound 2
from the nosN-knockout mutant of a nosiheptide high-
producing strain (Methods and Figure S1–S3 in the Support-
ing Information), and performed the assays with 2 and the
other required components. Again, no methylated product
was observed in the assay mixtures, suggesting that 2 is likely
not the NosN substrate but rather an off-pathway product
produced by the mutant strain.
Because the indolic acid moiety in nosiheptide is attached
to a cysteine thiol as a thioester (Figure 1A), it is possible that
MIA is first converted into an activated form before being
methylated by NosN and incorporated into the nosiheptide
scaffold.[4] Thioester linkage to the cysteamine group of
a phosphopantetheinyl cofactor is arguably the most common
strategy used by nature for the activation of a carboxylate,
and in biochemical studies, N-acetylcysteamine (SNAC)
thioesters (Figure 2B) have been widely used as surrogates
of phosphopantetheinylated substrates.[12] We therefore syn-
thesized SNAC thioester 4 (Figure 2B) and used it as
a potential substrate in the NosN assays. In this analysis,
a product with a protonated molecular ion at m/z = 291.1161
in LC–HRMS analysis was observed (Figure 2C and Fig-
ure S5), which is absent in all the three negative control
reactions (Figure S6). The suggested molecule formula
C15H18N2O2S ([M+H]+ calc. 291.1167, 2.0 ppm error) is
consistent with the methylated product 5 (Figure 2B), the
identity of which was further supported by comparative
HRMS/MS analysis (Figure S7). To further confirm the
production of 5 in the reaction, we treated the deproteinized
fraction of the assay mixture with NaOH, which leads to
hydrolysis of any thioesters, and the resulting mixture was
subjected to LC–HRMS analysis. As expected, a product
consistent with 3,4-dimethyl-2-indolic acid (6) was observed
([MÀH]À calc. 188.0711, obs. 188.0710, 0.5 ppm error), and its
identity was further supported by comparison with two
synthetic dimethylindolic acid isomers (Figure S8). These
results clearly indicate that NosN is a novel radical SAM
methyltransferase that is responsible for appending a methyl
group onto C4 of the indolyl moiety in nosiheptide biosyn-
thesis.
We next synthesized S-adenosyl-l-[methyl-2H3]-methio-
nine (d3-SAM) and used it in the NosN assay. LC–HRMS
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analysis of the resulting mixture showed that the methylated
product exhibits a protonated molecular ion at m/z =
293.1282 (3.7 ppm error for molecular formula of
edge, there is no biochemical precedent for the participation
of MTA in a methylation reaction. We ran the NosN assay
with MTA, d3-SAM, and the other required components, and
both 5 and 5d were observed in the reaction (Figure S9). This
analysis supports the idea that MTA is a direct methyl donor
in NosN catalysis, which raises the possibility that 5’-
thioadenosine (tAdoH; Figure 2D) is released as a co-
product in the reaction. Re-examination of the LC–HRMS
data showed that tAdoH was indeed produced in the assay
(Figure 2E), and the identity of this compound was supported
by coelution with the synthetic standard (Figure 2E) and by
comparative HRMS/MS analysis (Figure S10). Time course
analysis of the reaction mixture showed that tAdoH and 5
were produced in a roughly 1:1 ratio (Figure S11), thus
suggesting that tAdoH is likely an authentic co-product in the
NosN reaction.
a
C15H18D2N2O2S; Figure 2C), thus suggesting that the product
(5d) contains a methyl group with two deuterium atoms
(Figure 2B). This observation is reminiscent of the reactions
catalyzed by the RNA methyltransferases RlmN and Cfr, two
class A RSMTs that catalyze reactions involving methylated
cysteine residues.[13] To test whether NosN uses a similar
catalytic strategy involving a protein-bound methyl group, we
performed an assay under single-turnover conditions with
a limited amount of d3-SAM. LC–HRMS analysis of the assay
mixture showed that, unlike the single-turnover reactions
catalyzed by RlmN and Cfr, in which exogenous SAM is not
the source of the appended methyl group,[13b] only 5d was
produced in this assay, and the unlabeled product 5 was not
observed. This analysis suggests that NosN is different from
RlmN and Cfr and does not use a protein-bound methyl
group. To further validate this proposal, Cys50 and Cys108,
the two Cys residues besides those required for [4Fe-4S]
binding, were each replaced by an Ala, and the resulting
mutant enzymes were reconstituted and tested in the same
assay used for the wild type NosN. The results showed that
both mutants are able to produce 5 from 4, thus further
excluding the possibility that NosN employs a methylated Cys
residue in catalysis.
The results presented above suggest that NosN employs
an unprecedented mechanism for indole C4 methylation
(Figure 3). During catalysis, the enzyme converts one SAM
It has been shown that in the catalytic reactions of the
methylthioltransferases MiaB and RimO, the methyl group is
initially attached to the [4Fe-4S] cluster of the enzymes, and
exogenous methanethiol can serve as the methyl source in the
reaction.[14] However, in an assay with d3-SAM, NosN, and the
other required components in the presence of methanethiol,
unlabeled product was not produced in the reaction. This
analysis excludes the possibility that NosN uses a strategy
similar to MiaB and RimO in placing the methyl group on the
[4Fe-4S] cluster before its transfer to the substrate.
We next performed a detailed HPLC analysis to track the
fate of SAM in NosN catalysis. We noted that although the
reaction produced a substantial amount of dAdoH, the yield
of S-adenosylhomocysteine (SAH) is low and similar to the
levels in negative control reactions where methylation did not
occur (Figure 2A), thus suggesting that SAH is likely not
a co-product in NosN catalysis. This observation is in contrast
to all the SAM-dependent methyltransferases that have been
identified thus far, whether they use SN2 or radical-based
mechanisms. Unexpectedly, we found that 5’-methylthioade-
nosine (MTA) was produced at high yields in the reaction
(Figure 2A). Although this compound was also present in the
control assay with boiled enzyme, the yields of MTA in the
assays with the reconstituted NosN were apparently higher
(Figure 2A), thus indicating that MTA could possibly be
a relevant intermediate in the catalysis. The yield of MTA in
the absence of the reducing system (flavodoxin, flavodoxin
reductase, and NADPH) is similar to that for the normal
reaction, thus suggesting that MTA production is independent
of SAM reductive cleavage.
Figure 3. Proposed mechanism for the NosN-catalyzed methylation
reaction. In the enzyme active site, one SAM is converted into MTA
and the other is reductively cleaved to produce a dAdo radical. The
yield of homoserine lactone (HSL), the product of SAM degradation, is
low in the reaction, and owing to its lability under the assay
conditions, whether it is a co-product or not remains to be further
validated. DFT calculations on the model compounds suggests that for
À
the radical anion 9, heterolytic cleavage of its C S bond in pathway II
is energetically favored over homolytic cleavage (pathway I, the model
products of this pathway were used as the reference zero energy).
Both geometry optimization and energy were calculated at the B3LYP/
6-311+G(2d,p)/SMD(H2O) level.
molecule into MTA as a direct methyl donor and a second
SAM into a dAdo radical. The dAdo radical then abstracts
a hydrogen atom from the MTA methyl group, and the
resulting methylene radical 7 adds to the indolyl substrate at
C4 to form key radical intermediate 8. As in the mechanism
proposed for the RlmN-catalyzed reaction,[2c] 8 is likely
MTA is a known co-product in the biosynthesis of
polyamine, ethylene, and diphthamide,[15] and also a product
of SAM degradation.[16] However, to the best of our knowl-
À
deprotonated to produce radical anion 9, which undergoes C
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S bond cleavage to eliminate the thioadenosine moiety
(Figure 3). Density functional theory (DFT) calculations
and orbital analysis of the model compound showed that
the highest occupied molecular orbital (HOMO a) of 8 has
apparent spin density on the C4 hydrogen, while this spin
density decreases in the corresponding lowest unoccupied
molecular orbital (LUMO b, electron hole; Figure S12). This
observation suggests that the C4 hydrogen of 8 could possibly
be prone to deprotonation, which is consistent with our
proposal (Figure 3).
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Our study presents a new paradigm for SAM-dependent
methyltransferases, in which MTA is used as the direct source
of the methyl group. Unlike class A RSMTs, which employ
a ping-pong mechanism in which two SAM molecules
sequentially bind to the enzyme,[13] NosN likely binds two
SAMs simultaneously in the active site, where one SAM is
converted into MTA and the second into an dAdo radical to
initiate the radical-based reaction. The observation of two
SAM molecules in the crystal structure of the NosN
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Acknowledgements
This work is supported in part by grants from National
Natural Science Foundation of China (1500028 and 31670060)
and from the National Key Research and Development
Program (2016 Y F A0501302). Q.Z. would also like to thank
the Thousand Talents Program for support.
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Keywords: biosynthesis · methyltransferases · natural products ·
radical SAM enzymes · thiopeptides
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Manuscript received: October 11, 2016
Revised: December 15, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Biosynthesis
W. Ding, Y. Li, J. Zhao, X. Ji, T. Mo,
H. Qianzhu, T. Tu, Z. Deng, Y. Yu, F. Chen,
Q. Zhang*
&&& — &&&
The Catalytic Mechanism of the Class C
radical S-Adenosylmethionine
Methyltransferase NosN
Play something new, SAM: Mechanistic
investigation of the class C radical S-
adenosylmethionine (SAM) methyltrans-
ferase NosN showed that, in contrast to
all known SAM-dependent methyltrans-
ferases, NosN does not produce S-ade-
nosylhomocysteine as a co-product.
Instead, NosN converts SAM into 5’-
methylthioadenosine as a direct methyl
donor, employing a radical-based mech-
anism for methylation and releasing 5’-
thioadenosine as a co-product.
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!