NosN, a Radical S-Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide

Article abstract of DOI:10.1021/jacs.7b08492

Nosiheptide, a member of the e series of macrocyclic thiopeptide natural products, contains a side-ring system composed of a 3,4-dimethylindolic acid (DMIA) moiety connected to Glu6 and Cys8 of the thiopeptide backbone via ester and thioester linkages, re

Full text of DOI:10.1021/jacs.7b08492

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Article
The Radical S-Adenosylmethionine Methylase NosN Catalyzes
both C1 Transfer and Formation of the Ester Linkage of the
Side-Ring System during the Biosynthesis of Nosiheptide
Joseph W. LaMattina, Bo Wang, Edward D. Badding, Lauren K. Gadsby, Tyler Grove, and Squire J. Booker
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08492 • Publication Date (Web): 17 Oct 2017
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Journal of the American Chemical Society
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The Radical S-Adenosylmethionine Methylase NosN Catalyzes both
C1 Transfer and Formation of the Ester Linkage of the Side-Ring
System during the Biosynthesis of Nosiheptide
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Joseph W. LaMattina‡, Bo Wang‡, Edward D. Badding‡, Lauren K. Gadsby§, Tyler L. Grove‡,†, and
Squire J. Booker*,‡,§,#
Departments of ‡Chemistry and §Biochemistry and Molecular Biology, and the #Howard Hughes Medical Institute, The
Pennsylvania State University, University Park, Pennsylvania 16802, USA.
ABSTRACT: Nosiheptide, a member of the e series of macrocyclic thiopeptide natural products, contains a sideꢀring system comꢀ
posed of a 3,4ꢀdimethylindolic acid (DMIA) moiety connected to Glu6 and Cys8 of the thiopeptide backbone via ester and thioester
linkages, respectively. Herein, we show that NosN, a predicted class C radical Sꢀadenosylmethionine (SAM) methylase, catalyzes
both the transfer of a C1 unit from SAM to 3ꢀmethylindolic acid linked to Cys8 of a synthetic substrate surrogate as well as the
formation of the ester linkage between Glu6 and the nascent C4 methylene moiety of DMIA. In contrast to previous studies that
indicated that 5'ꢀmethylthioadenosine is the immediate methyl donor in the reaction, in our studies SAM itself plays this role, givꢀ
ing rise to Sꢀadenosylhomocysteine as a coꢀproduct of the reaction.
Nosiheptide (NOS) is a thiopeptide natural product that disꢀ
plays potent in vitro activity against a number of bacterial
pathogens of urgent clinical relevance, including methicillinꢀ
resistant Staphylococcus aureus, penicillinꢀresistant Strepto-
coccus pneumoniae, and vancomycinꢀresistantꢀenterococci.1ꢀ3
It is composed of a core peptide macrocycle that contains mulꢀ
tiple thiazole rings, dehydrated serine and dehydrated threoꢀ
nine amino acids, and
a central tetraꢀsubstituted 3ꢀ
hydroxypyridine ring. In addition to several other postꢀ
translational modifications, NOS contains a sideꢀring system
formed via a 3,4ꢀdimethylꢀ2ꢀindolic acid (DMIA) bridge that
connects the 4ꢀmethyl group of DMIA to Glu6 of the peptide
framework and the carboxylic acid group of DMIA to Cys8.3
Despite its potent in vitro activity, NOS and other similar thiꢀ
opeptide natural products suffer from poor solubility and gasꢀ
trointestinal absorption, limiting their use in the clinic.3
In pioneering studies by Wen Liu and coworkers, the gene
cluster for the biosynthesis of NOS was identified (nosAꢀnosP)
and the encoded gene products were assigned preliminary
functions based on amino acid similarity with proteins of
known functions and knockꢀout studies in Streptomyces actu-
osus, one of the producers of NOS.1 The thiopeptide is derived
from a ribosomally synthesized precursor peptide of 50 amino
acids (aa), which is the product of the nosM gene. The first 37
aa of the precursor peptide compose a leader sequence, which
is used primarily as a recognition motif for some of the enꢀ
zymes involved in NOS biosynthesis, while the last 13 aa
compose the structural peptide and are incorporated into the
final product. A model was put forward, wherein NosD, E, F,
G, H, M, and O are responsible for generating the core thioꢀ
peptide, while NosA, B, and C are responsible for additional
postꢀtranslational modifications to the core thiopeptide.1 NosI,
K, L, and N were assigned functions related to generating the
DMIA moiety and attaching it to the structural peptide, while
Figure 1. Proposed pathway for NOS sideꢀring formation.
NosP was suggested to be a regulator of the biosynthetic gene
cluster. NosJ, however, was not assigned a function. NosL, a
member of the radical Sꢀadenosylmethionine (SAM) superꢀ
family, has been shown to catalyze the first step in the forꢀ
mation of DMIA, which is the conversion of
Lꢀtryptophan into
3ꢀmethylꢀ2ꢀindolic acid (MIA), while NosN, annotated as a
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class C radical SAM (RS) methylase, was predicted to append
details).12 Purified NosN exhibits features that are consistent
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a methyl group to C4 of MIA or an acylated derivative of it to
form DMIA.4 NosK was predicted to transfer MIA or DMIA
to Cys8 of the structural peptide, and the 4ꢀmethyl group was
proposed to be hydroxylated by one of the two annotated cytoꢀ
chrome P450ꢀlike enzymes. Lastly, NosI was proposed to genꢀ
erate the Glu6 ester linkage of the sideꢀring system by adenylꢀ
ating Glu6 and catalyzing the attack of the C4ꢀhydroxyl group
of 3ꢀmethylꢀ4ꢀhydroxymethylꢀ2ꢀindolic acid onto the resulting
Glu6 acylꢀphosphate (Figure 1).1
with the presence of a [4Fe–4S] cluster, with a maximum abs
orbance at 280 nm and a broad feature at 410 nm (Figure S2,
dashed spectrum). The asꢀisolated enzyme contains 4.6 ± 0.2
iron ions and 3.2 ± 0.7 sulfide ions per polypeptide. Upon
reconstitution and subsequent purification by gelꢀfiltration
chromatography, NosN contains 3.7 ± 0.1 iron ions and 5.4 ±
0.4 sulfide ions per protein (Figure S2, solid spectrum).
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NosN Recognizes MIA-SNAC as a Substrate
Our recent studies,5 and subsequent studies by Ding et al.,6
have led to a modification of the above working hypothesis.
We showed that NosJ is an acyl carrier protein (ACP), and that
NosI catalyzes the ATPꢀdependent activation of MIA to MIAꢀ
AMP and the transfer of MIA to NosJ. NosK then transfers
MIA from MIAꢀNosJ to a conserved seryl residue (S102) on
itself, presumably for transfer to NosM during some stage in
the maturation of NOS.5 When NosK catalyzes this step has
not been determined; however, the structure of the protein,
which we solved to 2.3 Å resolution, reveals that Ser102 is
part of a SerꢀGluꢀHis catalytic triad located at the bottom of an
open cleft that is large enough to accommodate the structural
peptide.5 Our redetermination of the role of NosI in NOS bioꢀ
synthesis and the recent findings that both annotated P450ꢀlike
proteins in the NOS biosynthetic gene cluster have been asꢀ
signed specific functions7, suggest that the in vivo function of
NosN is not that of a methylase, but that it catalyzes both the
transfer of a C1 unit from SAM and the subsequent formation
of the ester linkage of the sideꢀring system by nucleophilic
addition (Figure 1).
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The identity and availability of the substrate for NosN has
hindered detailed mechanistic studies of its reaction. Recent
reports, however, indicate that NosN can convert the Nꢀ
acetylcysteamine (SNAC) derivative of MIA to DMIAꢀ
SNAC.8 SNAC analogs have been employed to imitate the
structure of the 4'ꢀphosphopanthetheine prosthetic group of
acyl carrier proteins during polyketide biosynthesis.13 Given
its ability to recognize MIAꢀSNAC, it seemed likely that the
true substrate for NosN might be MIA attached to NosJ or
MIA attached to Cys8 of the structural peptide at some stage
of nosiheptide maturation.6 As shown in Figure S3, NosN is
unable to convert MIAꢀNosJ to the DMIA derivative in our
hands, although this ability has recently been reported by Ding
et al.6 Although 5'ꢀdeoxyadenosine (5'ꢀdA) is produced, neiꢀ
ther MTA nor SAH is produced as a coꢀproduct, indicating
that the transfer of a C1 unit has not taken place. However, as
shown previously, NosN is able to convert MIAꢀSNAC to
DMIAꢀSNAC when incubated under turnover conditions
(SAM plus dithionite), as evidenced by the production of a
species exhibiting a massꢀtoꢀcharge ratio (m/z) of 291, and
which can be fragmented in the MS to daughter ions exhibitꢀ
ing m/z values of 144 and 172 (Figure S4, panel A, black
trace) (Figure S5, panel A, black trace).8 Interestingly, howꢀ
Recent studies from the Zhang laboratory detailed the first
in vitro characterization of the NosN reaction.8 As a member
of the RS superfamily of enzymes, NosN is believed to use a
5'ꢀdeoxyadenosyl 5'ꢀradical (5'ꢀdA•), derived from a reductive
cleavage of SAM, to initiate radicalꢀbased catalysis.9ꢀ10 Howꢀ
ever, exactly when NosN performs its reaction during NOS
maturation is unknown, which has limited mechanistic studies
of the enzyme. Although nosN knockout mutants of S. actu-
osus produce what appears to be a nearꢀfully elaborated NOS
that lacks the C4 methyl group of DMIA, and therefore the
ester linkage of the sideꢀring system, this molecule is not a
substrate for NosN.8 Moreover, reports from the Liu laboratoꢀ
ry have suggested that many of the postꢀtranslational modifiꢀ
cations to NOS do not take place after the core thiopeptide has
been synthesized, but are installed at various steps during the
biosynthesis of the core thiopeptide.11
In this work, we provide experimental evidence that NosN
catalyzes both carbon transfer from SAM and formation of the
ester linkage between Glu6 and the appended C4 carbon unit
of DMIA. When a small synthetic substrate containing both
Glu6 and Cys8 linked to MIA is used, we find that NosN cataꢀ
lyzes the capture of an intermediate containing a reactive C4
exocyclic methylene group by Glu6, which forms the ester
linkage and completes the biosynthesis of the sideꢀring system
of NOS.
Figure 2. MS MRM chromatogram of the 170 m/z fragment
from the 289 m/z parent ion produced in the NosN reaction
with 50 ꢁM enzyme, 1 mM SAM, 1 mM MIAꢀSNAC, 1 mM
sodium dithionite, and quenched with H2SO4. Traces are of
assays incubated for 90 min without MIAꢀSNAC (i), without
dithionite (ii), without SAM (iii), without NosN (iv), everyꢀ
thing present after 1 min (v) and after 90 min (vi). The peak in
trace vi marked by the asterisk is an artifact that arises under
these chromatography conditions.
RESULTS
Over-production and purification of NosN
NosN containing an Nꢀterminal hexahistidine tag was puriꢀ
fied to ≥95% homogeneity (Figure S1, panels A and B) using
methods that have already been described (See SI for
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acid (4OHMMIA). After neutralizing the reaction and separatꢀ
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ing the small molecules on a C18 column with MRM monitorꢀ
ing, formation of the hydroxymethyl adduct of MMIA (204
m/z) is observed (Figure 3, Panel iii). An additional peak is
also observed at 4.2 min at a 1:1 ratio, based on the absorption
at 300 nm, and has similar mass and fragment transitions. We
suggest that this peak may represent C1 addition at another
carbon of MIAꢀSNAC as a result of the smaller substrate anaꢀ
log hampering specificity for C1 transfer by NosN.
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Use of [methyl-2H3]-SAM results in deuterium loss during
NosN turnover
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Reactions were conducted with [methylꢀ2H3]ꢀSAM to conꢀ
firm that the added C1 unit in MMIAꢀSNAC ultimately deꢀ
rives from SAM. When the mass spectrum of MMIAꢀSNAC
produced in this reaction is compared to that of the reaction
containing SAM at natural abundance, a mass increase of two
is observed in the parent ion, which is consistent with the loss
of one deuterium atom from SAM during the transfer of the
Figure 3. Derivatization of MMIAꢀSNAC to 4ꢀ
hydroxymethylꢀ3ꢀmethylꢀ2ꢀindolic acid (4OHMMIA).
Samples were monitored by UVꢀabsorption at 300 nm
(blue traces) and the MRM transition of 204ꢀ160 m/z
for 4OHMMIA (black dotted traces). (Panel i) Standards
of 4OHMMIA and MIA were separated by UPLC. (Panꢀ
els ii and iii) NaOHꢀquenched NosN assays with 200 µM
enzyme and using 1 mM MIAꢀSNAC as the substrate
were analyzed by the developed method and compared at
t=1 min and 120 min, respectively. (Panel iv) NaOHꢀ
quenched NosN assay at 120 min using 1 mM MIAꢀ
t3NosM as a substrate. The shift in elution times between
the solid blue traces and the dotted black traces are due to
different distances between the end of the column and the
respective detector. Peaks under the asterisk represent an
unknown product formed during the assay.
ever, in our NosN assays, a product that elutes at 3.5 min by
HPLC exhibits an m/z of 289 and gives a daughter ion of 170
m/z during MS/MS fragmentation (Figure 2, trace vi) (Figure
S5, panel A, red trace). If either SAM, dithionite, NosN, or
MIAꢀSNAC is omitted from the reaction mixture, these prodꢀ
ucts are not observed (Figure 2, traces i-iv). Unlike the subꢀ
strate, which exhibits a UVꢀvis spectrum with an absorbance
maximum at 320 nm, the UVꢀvisible spectrum of the 289 m/z
species has an absorbance maximum around 300 nm (Figure
S5, panel B). The mass, UVꢀvis spectrum, and elution profile
suggests that the observed species may be 4ꢀmethyleneꢀ3ꢀ
methylꢀindolic acidꢀSNAC (MMIAꢀSNAC) that is protonated
at N1. This conclusion is consistent with this molecule’s alꢀ
tered conjugation and reduced retention time on a C18 UPLC
column, which, in turn is likely due to the molecule’s inherent
positive charge, giving rise to its [M+] charge state. The peak
that elutes at 4 min (Figure 2, trace vi) is an artifact of the
chromatography conditions. When the gradient is shallower,
this peak is not present.
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Figure 4. (Panels A and B) Mass spectrum of MMIAꢀ
SNAC and 5'ꢀdA, respectively, produced when 1 mM
SAM is used in the NosN reaction (black traces) and
when 1 mM [methylꢀ2H3]ꢀSAM is used in the reaction
(red traces) with 50 ꢁM NosN.
C1 unit (Figure 4, panel A). Correspondingly, when 5'ꢀdA
(m/z 252.1) produced by the reaction containing [methylꢀ2H3]ꢀ
SAM is monitored by MS, enrichment of the A+1 isotope (m/z
253.1) is observed, indicating incorporation of deuterium from
the methyl group of SAM (Figure 4, panel B). The concentraꢀ
tions of 5'ꢀdA and Sꢀadenosylhomocysteine (SAH) were also
quantified as a function of time, and their progress curves at
varying concentrations of NosN were compared (Figure 5,
panels A and B). The progress curves for SAH and 5'ꢀdA
were fitted to Frieden’s equation (Eq. 1) to assess the preꢀ
steady state burst, which we attribute to a slow release of
MMIAꢀSNAC.14 DMIAꢀSNAC is also observed at a low inꢀ
tensity (Figure S4), which we believe is an offꢀpathway prodꢀ
Derivatization of MMIA-SNAC to 4-hydroxymethyl-3-methyl-
2-indolic acid
To provide further support for formation of a NosNꢀ
catalyzed MMIA intermediate when using MIAꢀSNAC as a
substrate in the reaction, reactions were quenched in a final
concentration of 2 M NaOH to promote the nucleophilic addiꢀ
tion of hydroxide ion onto the electrophilic exocyclic methꢀ
ylene carbon, affording 4ꢀ(hydroxymethyl)ꢀ3ꢀmethylꢀ2ꢀindolic
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appears to act as a modest inhibitor of the reaction (Figure 6).
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Moreover, in time courses of the NosN reaction, no MTA is
observed that is above its background levels in our preparaꢀ
tions of SAM.
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Use of a peptide mimic to assess the catalytic mechanism of
NosN
The observation that NosN catalyzes only one turnover at
most with the MIAꢀSNAC substrate, coupled with the producꢀ
tion of MMIAꢀSNAC that is slowly converted to DMIAꢀ
SNAC, suggests the slow release of an intermediate species.
We posited that with the natural substrate, NosN might direct
Glu6 to react with the C4 exocyclic methylene group of
MMIAꢀSNAC, thereby generating the ester linkage and comꢀ
pleting the formation of the sideꢀring system. Therefore, we
synthesized a more natural mimic of the NosN substrate,
termed MIAꢀt3NosM. MIAꢀt3NosM derives from residues 42ꢀ
46 of NosM, and contains the three thiazole rings that surꢀ
round Glu6 and Cys8, as well as the thioester linkage between
Cys8 and MIA. Our choice of this substrate was dictated by
ease of synthesis—not having to incorporate dehydrated amiꢀ
no acids—while maximizing the peptide length. MIAꢀt3NosM
exhibits an m/z of 684 and elutes at 5 min by UPLC using a
C18 column under the described conditions (Figure S6, panel
B, trace i). When MIAꢀt3NosM is incubated with SAM, dithiꢀ
onite and NosN, two peaks, each exhibiting m/z 696, are obꢀ
served to increase with time (Figure 7, panel A, black trace)
(Figure S6, panel B, trace iv) (Figure S6, panel C, black
trace). MS/MS analysis of the 696 m/z ion produces promiꢀ
nent daughter ions with m/z values of 170 and 188, the latter
of which is consistent with the addition of a hydroxyl group to
the exocyclic methylene on the MMIA moiety (Figure 7,
panel B, black trace) (Figure S6, panel D, black trace).
Correspondingly, when [methylꢀ13C]ꢀSAM is used in reacꢀ
tions, a dominant ion of m/z 697 is observed, indicating 13C
incorporation into the molecule (Figure S6, panel C, red
trace). The observation of an increase in mass of one indicates
that the carbon unit from SAM is associated with the fragꢀ
ment, which we assign as an MIA derivative (Figure S6, pan-
el D, red trace). Although NosN catalyzes considerable aborꢀ
tive cleavage of SAM in the presence of MIAꢀt3NosM (Figure
S7), the formation of MIAꢀt3NosM containing the sideꢀring
(NosMSR) system was monitored by MRM (m/z 696), and the
progress curve was compared to that for the formation of SAH
(Figure 7, panel A, dotted and solid lines, respectively).
MTA is also observed; however, its normalized intensity does
not change with time (Figure S7, panel C).
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Figure 5. (Panels A and B) Progress curves for formation of
5'ꢀdA and SAH in assays with 10, 20, 30, 40, and 50 µM
NosN (black, magenta, blue, green, and orange traces, reꢀ
spectively). All assays contain 1 mM MIAꢀSNAC, 1 mM
SAM, and 2 mM sodium dithionite.
uct, as will be detailed below.
MTA is not the direct methyl donor for the NosN reaction
Intriguingly, previous studies by Zhang and coworkers indiꢀ
cated that MTA is the immediate methyl donor in the NosN
reaction.8 We probed this possibility further by performing
reactions with [methylꢀ13C]ꢀSAM in the presence of MTA at
natural abundance and monitoring MMIAꢀSNAC formation.
Even upon preꢀincubating MTA with NosN and initiating the
reactions by adding [methylꢀ13C]ꢀSAM, only ~4% of the
MMIAꢀSNAC produced is at natural abundance, while MTA
Glu6 of NOS traps the exocyclic methylene intermediate
To test the possibility that Glu6 attacks the exocyclic methꢀ
ylene group of MMIA during formation of NosMSR, a variant
of the MIAꢀt3ꢀNosM substrate containing a Glu6→Ala substiꢀ
tution (MIAꢀt3NosMglu→ala) was synthesized (Figure S8).
When this substrate is incubated with NosN under turnover
conditions, a dominant signal exhibiting m/z of 640 is obꢀ
served, which is consistent with incorporation of one methyl
group into the MIAꢀt3NosMglu→ala peptide (Figure 7, panel A,
blue trace). The fragmentation pattern of this product (640
m/z) contains a major species at m/z 172, which is consistent
with the fragmentation patterns of the methylated products
DMIAꢀSNAC and DMIAꢀt3NosM (Figure 7, panel B, blue
Figure 6. Progress curves for C1 transfer to MIAꢀSNAC
using either [methylꢀ13C]ꢀSAM or MTA as the C1 donor.
[methylꢀ13C]ꢀSAM was added to a 500 µM final concentraꢀ
tion in the presence of 0, 250, and 500 µM MTA (black,
red, and blue traces, respectively). Formation of 13Cꢀlabeled
MMIA (closed circles) was compared to that of unlabeled
MMIA (open circles).
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Figure 7. (Panel A) Chromatogram displaying the SIM of the dominant product formed when 1 mM MIAꢀt3NosM (black),
MIAꢀt3NosMglu ala (blue), and [γꢀcarboxyꢀ18O]ꢀMIAꢀt3NosM (red) are used in the NosN assay with 50 ꢁM enzyme. (Panel
ꢀ
B) MS/MS spectrum of the corresponding parent ion from panel A.
trace). Furthermore, the 188 m/z product ion—produced when
notated as a class C RS methylase. Class C RS methylases
belong to the HemN family of RS enzymes, which are beꢀ
lieved to use two simultaneously bound molecules of SAM to
methylate sp2ꢀhybridized carbon centers. The in vitro characꢀ
terization of class C methylases has been sluggish, however,
because of the complex substrates on which they operate. Our
recent studies, which led to a reꢀevaluation of the proposed
NOS biosynthetic pathway, suggest that the substrate for
NosN contains the core thiopeptide at some stage during its
maturation, but after incorporation of the thioester linkage to
MIA and before NosOꢀcatalyzed formation of the central hetꢀ
erocycle.5
fragmenting the NosMSR—is not observed from this product,
suggesting that the 188 m/z ion is a result of having Glu6 in
the peptide (Figure S8). To further confirm the origin of the
hydroxyl group in the 188 m/z product ion, MIAꢀt3NosM conꢀ
taining a [γ
-carboxyꢀ18O]ꢀglutamyl residue was prepared (Fig-
ure S9) for use in NosN reactions. The resulting compound is
a mixture of two species: one (~37%) contains two incorpoꢀ
rated 18O atoms, while the second (~43%) contains only one
incorporated 18O atom. When the 18Oꢀcontaining MIAꢀt3ꢀ
NosM is used in a NosN reaction, a mass shift of either 2 or 4
is observed in the NosMSR, indicating retention of the isotopic
label in the glutamyl residue during turnover (Figure 7, panel
A, red trace). More importantly, during MS/MS analysis of
the 700 m/z parent ion, a new peak of 190 m/z is observed,
which is consistent with incorporation of one 18O atom into the
188 m/z peak from NosMSR, (Figure 7, panel B, red trace),
unambiguously assigning Glu6 as the source of the hydroxyl
substituent, and confirming the presence of an ester linkage to
the C4 methyl group of DMIA. After base hydrolysis of
NosMSR, and analysis by LCꢀMS, we observe production of 4ꢀ
(hydroxymethyl)ꢀ3ꢀmethylꢀ2ꢀindolic acid at a 9:1 ratio comꢀ
pared to the alternative product made during NosN assays with
MIAꢀSNAC (Figure 3, panel iii, blue trace), indicating that
with a more physiological substrate there is better site speciꢀ
ficity to the C4 position of the MIA moiety.
Recently, the Zhang laboratory described the first in vitro
characterization of NosN and reported some intriguing obserꢀ
vations.8 First, they showed that NosN can use MIAꢀSNAC as
a substrate, which it converts to DMIAꢀSNAC. Second, they
showed that NosN first converts one molecule of SAM to
MTA, which then becomes the direct methyl donor in the
NosN reaction. They propose a mechanism whereby one molꢀ
ecule of SAM is reductively cleaved to generate a 5’ꢀdA•,
which then abstracts a hydrogen atom from MTA. The resultꢀ
ing methylene radical then adds to C4 of MIA to give a subꢀ
strate radical intermediate. Upon loss of the C4 proton, the
resulting radical anion species eliminates 5’ꢀthioadenosine (5’ꢀ
tA) to afford a substrate radical intermediate containing a C4
exocyclic methylene group. The addition of an electron and a
proton to this species affords the methylated product. More
recently, the Zhang laboratory also showed that NosJꢀMIA
also serves as a substrate for NosN, which it converts to NosJꢀ
DMIA.6
DISCUSSION
The formation of the sideꢀring system of NOS requires the
action of five dedicated proteins: NosI, NosJ, NosK, NosL,
and NosN. Two of these proteins, NosL and NosN, belong to
the RS superfamily of enzymes. NosL catalyzes a complex
rearrangement of tryptophan to give MIA, while NosN is anꢀ
In our studies herein, we also show that MIAꢀSNAC is a
substrate for NosN; however, in contrast to the observations
reported by the Zhang laboratory, we observe SAH as a
coproduct rather than 5’ꢀtA. Moreover, when we include MTA
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Figure 8. Proposed mechanism for the NosN reaction.
in reactions containing [methylꢀ13C]ꢀSAM, less than 4% of the
DMIA product contains an added C1 unit at natural abunꢀ
dance. The C1 unit that is transferred derives almost excluꢀ
sively from [methylꢀ13C]ꢀSAM. Moreover, product curves
show an initial burst of SAH production followed by a slower
steadyꢀstate phase of SAH production. Importantly, we obꢀ
serve the production of a species that we believe is an interꢀ
mediate in the reaction (MMIAꢀSNAC), and which we believe
contains an electrophilic C4 methylene group. This determinaꢀ
tion is based on the shift in its UVꢀvis spectrum from that of
MIA and DMIA, its mass, its elution profile, and the observaꢀ
tion that it is readily converted to 4ꢀhydroxymethylꢀ3ꢀMIAꢀ
SNAC upon treatment with base. MMIAꢀSNAC is slowly
converted to DMIAꢀSNAC in a reaction that we believe is off
pathway. Also, in contrast to observations by the Zhang laborꢀ
atory, NosJꢀMIA is not a substrate for NosN in our hands.
Although we observe the production of 5’ꢀdA, we do not obꢀ
serve production of SAH, MTA or NosJꢀDMIA. Our observaꢀ
tion of SAH rather than 5’ꢀtA formation is consistent with
recent studies on ChuW, which also resides in the HemN RS
subfamily. This enzyme is proposed to use a similar radical
addition mechanism during the anaerobic degradation of
heme, and also produces SAH rather than 5’ꢀtA as a coprodꢀ
uct.15
ster linkage to MIA as well as the three thiazole rings that
surround these two key amino acids. When we use this subꢀ
strate in NosN reactions, we observe formation of a species by
MS that fragments to give a C4 hydroxymethyl group on MIA.
If we use a similar substrate that differs only by an alanine
substitution for Glu6, the C4 hydroxymethylꢀMIA species is
not observed. Moreover, if we use a substrate containing a [γ-
carboxyꢀ18O]ꢀglutamyl residue, we observe a C4 hydroxymeꢀ
thylꢀMIA species that contains one atom of 18O.
In summary, NosN catalyzes the radicalꢀmediated transfer
of a C1 unit from SAM to C4 of MIA to generate SAH and a
reactive exocyclic intermediate that is captured by Glu6 of the
structural peptide to generate the ester linkage in the sideꢀring
system of NOS. Its ability to act as a methylase derives from a
slower, nonꢀenzymatic conversion to DMIA in the absence of
a good substrate. If a methyl moiety were to be appended to
C4 of MIA to give DMIA, there are no remaining unannotated
enzymes within the nos operon that could activate it for forꢀ
mation of the Glu6ꢀDMIA ester linkage, implying that NosJꢀ
MIA cannot be the true substrate for NosN.
EXPERIMENTAL PROCEDURES
Materials
Our working hypothesis for the NosN reaction is depicted in
Figure 8. A 5’ꢀdA•, obtained from the reductive cleavage of
SAM, is used to abstract a hydrogen atom from the methyl
moiety of another simultaneously bound molecule of SAM to
give a methylene radical. The methylene radical adds to C4 of
MIA that is connected in a thioester linkage to Cys8 of the
core peptide that has already been matured to contain thiazole
groups. The resulting delocalized substrate radical intermediꢀ
ate loses its C4 proton, which actuates the release of SAH to
afford a substrate radical intermediate containing a C4 exocyꢀ
clic methylene group. This species loses an electron back to
the FeꢀS cluster to give the electrophilic MMIA species, which
subsequently traps Glu6 to complete formation of the sideꢀring
system of NOS.
All commercial materials were used as received unless othꢀ
erwise noted. All DNAꢀmodifying enzymes and reagents used
were purchased from New England Biolabs. DNA isolation
kits were purchased from MacheryꢀNagel. PfuTurbo DNA
polymerase AD was purchased from Agilent Technologies
Inc. Sequencing grade trypsin was purchased from Promega
Corporation. Primers were purchased from Integrated DNA
Technologies. Deoxynucleotides were purchased from Denꢀ
ville Scientific Corporation. The expression vectors pETꢀ26b
and pETꢀ28a were purchased from EMD Millipore. Nꢀ(2ꢀ
hydroxyethyl)ꢀpiperazineꢀN′ꢀ(2ꢀethanesulfonic acid) (HEPES)
was purchased from Fisher Scientific. Imidazole was purꢀ
chased from J. T. Baker Chemical Co. Potassium chloride and
glycerol were purchased from EMD Chemicals. 2ꢀ
Mercaptoethanol and phenylmethanesulfonyl fluoride (PMSF)
were purchased from SigmaꢀAldrich. Isopropyl βꢀDꢀ1ꢀ
thiogalactopyranoside (IPTG) and dithiothreitol (DTT) were
To provide evidence for the mechanism in Figure 8, we
synthesized an artificial substrate comprised of residues 42ꢀ46
of the core peptide, which includes Glu6 and Cys8 in a thioeꢀ
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purchased from Gold Biotechnology. ATP (disodium salt) and 37 °C with shaking at 250 rpm. Several shake flasks of LB
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coenzyme A (trilithium salt) were purchased from Calbioꢀ
chem. 3ꢀMethylꢀ2ꢀindolic acid (MIA) was purchased from
Matrix Scientific. Talon metal affinity resin was acquired from
Clontech Laboratories Inc. Claritas PPT single element Fe
(1000 mg/L in 2% HNO3), used to prepare iron standards for
quantitative iron analysis, was obtained from SPEX CertiPrep
(Metuchen, NJ). Tetrahydrofuran and dichloromethane were
obtained from a JC Meyer solvent dispensing system and used
without further purification. Silia Flash 60® silica gel (230ꢀ
400 mesh) for flash chromatography was obtained from Siliꢀ
cycle Inc. All commercially available amino acid building
blocks were purchased from ChemꢀImpex International, Inc.
All other chemicals and materials were of the highest grade
available and were from SigmaꢀAldrich.
media were then inoculated with the overnight starter culture
to initiate growth (180 rpm at 37 °C). Expression of the genes
encoded on plasmid pDB1282 was induced at an OD600 of
~0.3 with 0.2% arabinose. Expression of the nosN gene was
subsequently induced with 0.5 mM IPTG at an OD600 of ~0.6,
and the flasks were immediately placed in an ice bath for 1 h.
Once cooled, the cultures were incubated overnight for 18 h at
18 °C with shaking at 180 rpm. The cells were harvested (~30
g), flashꢀfrozen in liquid nitrogen, and stored at ꢀ80 °C.
9
For purification, 30 g of frozen cell paste was resuspended
in 150 mL of lysis buffer (50 mM HEPES, pH 7.5, 300 mM
KCl, 10% (v/v) glycerol, and 10 mM BME) containing PMSF
(1 mM), DNase1 (100 µg mLꢀ1), and lysozyme (1 mg mLꢀ1).
Resuspended cells were incubated on ice and subjected to six
sonic bursts (40% output) on a QSonica instrument (housed in
a Coy anaerobic chamber) for 45 s each with 8ꢀmin intermitꢀ
tent pauses. The lysate was then centrifuged for 1 h at 50,000
× g at 4 °C. The resulting supernatant was loaded onto Talon
Co2+ resin equilibrated in the lysis buffer. The resin was
washed twice with 100 mL of the lysis buffer prior to elution
of Hisꢀtagged NosN with 50 mL of elution buffer (50 mM
HEPES, pH 7.5, 30 mMKCl, 250 mM imidazole, 10% (v/v)
glycerol, and 10 mM BME). The pooled eluate was concenꢀ
trated by ultracentrifugation using an Amicon Ultra centrifugal
filter unit with a 10ꢀkDa molecular weight cutoff membrane.
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General Methods
Sizeꢀexclusion chromatography was conducted using a
HiPrep 16/60 Sephadex Sꢀ200 column connected to an ÅKTA
fast protein liquid chromatography (FPLC) system in a Coy
anaerobic chamber (Grass Lake, MI). UVꢀvisible spectra were
recorded on a Cary 50 spectrometer from Varian (now Agilent
Technologies) using the WinUV software package to control
the instrument. High performance liquid chromatography
(HPLC) with detection by tandem mass spectrometry (LCꢀ
MS/MS) was conducted on an Agilent Technologies (Santa
Clara, CA) 1200 system coupled to an Agilent Technologies
6410 QQQ mass spectrometer. The system was operated with
the associated MassHunter software package, which was also
used for data collection and analysis. Highꢀresolution ESI
mass spectra were acquired in on a Waters LCT Premier inꢀ
strument at the Penn State Huck Institute of Life Sciences
proteomics and mass spectrometry core facility. DNA seꢀ
quencing was conducted at the Huck Institutes of the Life Sciꢀ
ences genomics core facility. NMR spectra were recorded on a
Bruker AVꢀ3ꢀHDꢀ500 instrument and calibrated using residual
solvent peaks as an internal reference. Multiplicities are recꢀ
orded as: s = singlet, d = doublet, t = triplet, dd = doublet of
doublets, m = multiplet, q = quartet, dt = doublet of triplets.
Amino acid analysis of NosN
A correction factor for the Bradford protein assay of 0.55
was obtained for NosN by amino acid analysis as previously
described.12 Asꢀisolated NosN was reconstituted with iron and
sulfide according to previously established procedures.12 The
stoichiometry of iron and sulfide per asꢀisolated and reconstiꢀ
tuted NosN polypeptide was determined by the methods of
Beinert—as described previously12—in concert with the corꢀ
rection factor for the Bradford protein assay.
NosN enzyme assays and quantification of products by LC-
MS/MS
All assays were conducted in a Coy anaerobic chamber
maintained under 95% N2 and 5% H2. The oxygen concentraꢀ
tion was maintained below 1 ppm via the use of palladium
catalysts. Typical assays were conducted in a volume of 200
ꢁL, and contained 50 mM HEPES (pH 7.5), 200 mMKCl, 5%
glycerol, 2 mM sodium dithionite, 50 ꢁM NosN, 1 mM MIAꢀ
Overproduction and purification of NosN
The gene encoding NosN was codonꢀoptimized for expresꢀ
sion in E. coli (sequence below) and synthesized by GeneArt
(Thermofisher Scientific). It was supplied in plasmid pMA
flanked between an NdeI site on its 5'ꢀterminus and an EcoRI
site on its 3'ꢀterminus. The nosN gene was excised from the
pMA plasmid by digestion with NdeI and EcoRI and then liꢀ
gated into pETꢀ28a that had been digested with the same reꢀ
striction enzymes and subsequently purified by gelꢀ
electrophoresis. This cloning strategy affords a NosN protein
that contains an Nꢀterminal hexahistidine (His6)ꢀtag that can
be exploited for protein purification by immobilized metal
affinity chromatography. After transformation of the ligation
mixture into E. coli DH5α, DNA was purified from several of
the colonies and subjected to sequencing. A plasmid containꢀ
ing the correct sequence (pET28aꢀnosN) was used to transform
E. coli BL21(DE3) for expression of the nosN gene under an
inducible T7 promoter.
SNAC or peptide mimic, and 100 ꢁM Lꢀtryptophan as an inꢀ
ternal standard. Reactions were initiated with 1 mM SAM, and
at various times, 15 ꢁL volumes were removed and added to
15 ꢁL of either 100 mM H2SO4 or 100% MeOH (for MIAꢀ
SNACꢀor peptide mimicꢀcontaining assays, respectively) to
quench the reaction. Quenched samples were centrifuged at
16,000 × g for 15 min before pipetting the supernatant into a
sample vial. Components of each quenched reaction were sepꢀ
arated on a Zorbax extendꢀC18 RRHT column (4.6 mm × 5
mm, 1.8 ꢁm particle size) equilibrated in 98% solvent A (0.1%
formic acid, pH 2.4) and 2% solvent B (100% acetonitrile).
For separation of reaction components in assays containing
MIAꢀSNAC, a linear gradient of solvent B from 2% to 10%
was applied from 0 to 0.5 min, which was followed by a linear
gradient to 50% from 0.5 to 2.5 min. Solvent B was then inꢀ
creased linearly to 100% from 2.5 to 3.5 min and held constant
E. coli Blꢀ21 (DE3) containing plasmid pDB1282 was transꢀ
formed with the pET28aꢀnosN. An LB media starter culture
was inoculated with a single colony and incubated overnight at
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from 3.5 to 4.5 min. Solvent B was then decreased linearly to applied from 1 to 4.5 min, which was followed by a linear
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2% from 4.5 to 5 min and held constant for 2.5 minutes to reꢀ
equilibrate the column before subsequent sample injections.
Products were detected using electrospray ionization in posiꢀ
tive mode (ESI+) and quantified with a multiple reaction moniꢀ
gradient to 100% from 4.5 to 6.5 min. Solvent B was held
constant from 6.5 to 7.5 min before being decreased linearly to
20% from 7.5 to 8.0 min and held constant for 2.5 minutes to
reꢀequilibrate the column before subsequent sample injections.
Products were detected using electrospray ionization in negaꢀ
tive mode (ESIꢀ) using an MRM method detecting unlabeled
and 13Cꢀlabeled 4ꢀhydroxymethylꢀ3ꢀmethylꢀ2ꢀindolic acid,
respectively.
toring (MRM) method for Lꢀtryptophan, SAH, 5′ꢀdA, MTA,
thioadenosine, MIAꢀSNAC, and MMIAꢀSNAC. For separaꢀ
tion and quantification of molecules from the assay containing
the peptide mimic, a linear gradient of solvent B from 2% to
10% was applied from 0 to 0.5 min, which was followed by a
linear gradient to 40% from 0.5 to 2.5 min. Solvent B was
increased linearly to 100% from 2.5 to 4.5 min and then held
constant from 4.5 to 5.5 min. Solvent B was then decreased
linearly to 2% from 5.5 to 6.0 min and held constant for 2.5
minutes to reꢀequilibrate the column before subsequent sample
injections. Products were detected using ESI+ and quantified
Progress curves for SAH and 5'ꢀdA formation from MIAꢀ
SNAC experiments were fit to Frieden’s equation using residꢀ
ual analysis (Eq. 1):
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ꢆ
ꢈ ꢆ
ꢉꢉ
ꢇ
ꢁ1 − ꢍꢈꢊ ꢎꢃ
(Eq. 1)
ꢋꢌꢉ
ꢁ ꢃ
ꢀ ꢂ = ꢄꢅꢅꢂ +
ꢊ
ꢋꢌꢉ
where t is time, vi is the steady initial velocity, vss is the steady
state velocity, and kobs is the rate of change between vi and vss.
with an MRM method for Lꢀtryptophan, SAH, 5′ꢀdA, MTA,
thioadenosine, MIAꢀt3NosM, and NosMSR, MIAꢀt3NosMglu
,
ala
and 18OꢀglutamateꢀNosMSR. Parameters for the MRM mꢀethꢀ
Progress curves resulting from the assay with MIAꢀt3NosM
were fit to a single exponential (Eq. 2):
od(s) are in Table S1.
Derivatization of MMIA was performed by quenching assay
timepoints with 2 M NaOH and incubating at 37 ºC overnight.
The baseꢀhydrolyzed mixture was neutralized with a molar
equivalent of HCl and a volume equivalent of 40 mM ammoꢀ
nium acetate. Components of the quenched reaction were sepꢀ
arated on a Zorbax extendꢀC18 RRHT column (4.6 mm × 5
mm, 1.8 ꢁm particle size) equilibrated in 80% solvent A (40
mM ammonium acetate, pH 6.0 and 5 % MeOH) and 20%
solvent B (100% MeOH). For separation of reaction compoꢀ
nents, a linear gradient of solvent B from 20% to 50% was
ꢀ ꢂ = ꢏꢍꢈꢁꢊ∙ꢎꢃ + ꢐꢑ
ꢁ ꢃ
(Eq. 2)
where A is the amplitude, k is the rate constant, t is time, and
yo is the initial concentration of 5'ꢀdA or SAH
The authors thank the Huck Institute for Life Sciences Geꢀ
nomics Core for DNA sequencing and the Proteomics and
Mass Spectrometry Core for smallꢀmolecule mass analysis.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
ABBREVIATIONS
4OHMMIA, hydroxymethylꢀ3ꢀmethylꢀ2ꢀindolic acid; 5’ꢀdA, 5’ꢀ
deoxyadenosine; aa, amino acids; DMIA, 3,4ꢀdimethylꢀ2ꢀindolic
acid; MIA, 3ꢀmethylꢀ2ꢀindolic acid; MRM, multiple reaction
monitoring; MS, mass spectrometry or mass spectrum; MTA,
methylthioadenosine; NOS, nosiheptide; RS, radical SAM; SAH,
Sꢀadenosylhomocysteine; SAM, Sꢀadenosylmethionine; SNAC,
Nꢀacetylcysteamine; SIM, singleꢀion monitoring
Optimized gene sequence for nosN, synthetic procedures, Table
S1, and Figures S1–S9 (PDF).
AUTHOR INFORMATION
Corresponding Author
* Squire J. Booker, squire@psu.edu
Present Addresses
REFERENCES
†Department of Biochemistry and Department of Physiology &
Biophysics, Albert Einstein college of Medicine, 1300 Morris
Park Av., Forchheimer building, Bronx, NY 10461
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ACS Chem Biol 2009, 4 (10), 855ꢀ64.
Author Contributions
This manuscript was written through contributions of all authors,
each of whom has given approval to the final version.
2.
Haste, N. M.; Thienphrapa, W.; Tran, D. N.;
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