S-nitroso proteome of Mycobacterium tuberculosis:
Enzymes of intermediary metabolism and
Kyu Y. Rhee*, Hediye Erdjument-Bromage†, Paul Tempst†‡, and Carl F. Nathan‡§¶?
*Division of International Medicine and Infectious Diseases, Department of Medicine, and§Department of Microbiology and Immunology, Weill Medical
College of Cornell University, New York, NY 10021;†Protein Center, Sloan–Kettering Institute, New York, NY 10021; and Programs in‡Molecular Biology
and¶Immunology, Weill Graduate School of Biomedical Sciences of Cornell University, New York, NY 10021
Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved November 22, 2004 (received for review
August 19, 2004)
The immune response to Mycobacterium tuberculosis (Mtb) in-
cludes expression of nitric oxide (NO) synthase (NOS)2, whose
products can kill Mtb in vitro with a molar potency greater than
that of many conventional antitubercular agents. However, the
targets of reactive nitrogen intermediates (RNIs) in Mtb are un-
known. One major action of RNIs is protein S-nitrosylation. Here,
we describe, to our knowledge, the first proteomic analysis of
S-nitrosylation in a whole organism after treating Mtb with bac-
tericidal concentrations of RNIs. The 29 S-nitroso proteins identi-
fied are all enzymes, mostly serving intermediary metabolism, lipid
metabolism, and?or antioxidant defense. Many are essential or
implicated in virulence, including defense against RNIs. For each of
two target enzymes tested, lipoamide dehydrogenase and myco-
bacterial proteasome ATPase, S-nitrosylation caused enzyme inhi-
bition. Moreover, endogenously biotinylated proteins were driven
into mixed disulfide complexes. Targeting of metabolic enzymes
and antioxidant defenses by means of protein S-nitrosylation and
mixed disulfide bonding may contribute to the antimycobacterial
actions of RNIs.
nitric oxide ? biotin ? lipoamide dehydrogenase ? mycobacterial
(Mtb) in mice and is expressed in macrophages of tuberculous
lesions in humans (1–5). The reactive nitrogen intermediate
(RNI) products of NOS2 exert potent time- and concentration-
dependent mycobactericidal activity in vitro (5). However, im-
munity to Mtb is rarely sterilizing and may be undermined by the
ability of Mtb to detoxify RNIs. If we knew which enzymes are
targeted by RNI to suppress or kill Mtb, we might be able to
inhibit these same enzymes with chemicals impervious to Mtb’s
Two major reactions of RNIs with cell constituents are
oxidation and S-nitrosylation (6–8). S-nitrosylation of specific
cysteine residues in microbial proteases and Zn-dependent
DNA-binding proteins accompanies the antimicrobial activities
of RNI against coxsackievirus, HIV, Leishmania, Plasmodium,
Trypanosoma, and Salmonella (7, 9–14). Despite the widespread
role of RNI as antimicrobial and regulatory molecules (15),
however, no systematic identification of S-nitroso proteins in any
organism has been described.
We adapted a biotin-switch method that selectively replaces
the NO moieties of target protein cysteine nitrosothiols with a
disulfide linkage to a biotin derivative by means of a 1,6-
diaminohexane spacer N-[6-(biotinamido)hexyl]-3?-(2?-pyri-
dyldithio)propionamide (biotin-HPDP) (16). This method per-
mits the detection of S-nitroso proteins by anti-biotin
immunoblotting and their purification by streptavidin affinity
chromatography. The work herein describes the identification of
the Mtb S-nitroso proteome in an effort to define one set of
itric oxide (NO) synthase (NOS)2 is critical for control of
acute and chronic infection by Mycobacterium tuberculosis
specific targets of RNIs that may help explain the potent
antimycobacterial properties of RNIs and identify potential
targets for the development of novel antitubercular drugs.
Mycobacterial Growth Conditions and Lysate Preparation. Mtb
H37Rv was grown in 7H9 broth with 10% oleic acid, albumin,
were washed twice in PBS (pH 7.2) containing 0.05% Tween 80
and resuspended in PBS (pH 5.5) supplemented with 10% 7H9
broth containing 10% OADC to an OD at 580 nm of 0.3
absorbance units. Bacteria remained fully viable as assessed by
colony-forming units and evenly dispersed over the course of 3
days under these conditions (data not shown) (17, 18). Bacteria
were exposed to a final concentration of 30 mM sodium nitrite
for 12 h at 37°C.
For macrophage infections, 4 ? 107bone marrow-derived
macrophages from wild-type or NOS2-deficient C57BL?6J mice
were incubated in the absence and presence of 100 units?ml
IFN-? with 4 ? 108colony-forming units of M. tuberculosis
H37Rv. After 4 h at 37°C, extracellular bacteria were removed
by washing twice with prewarmed PBS followed by replacement
of tissue culture media. Conditioned media sampled 16 h later
were assayed for nitrite accumulation by using the Griess assay
(19). Extracellular bacteria were removed again before lysis of
macrophages by washing twice with prewarmed PBS. Intracel-
lular bacteria were recovered by lysing the macrophages with
0.5% Triton X-100.
Bacterial lysates were maintained in the dark at or ?4°C.
Bacterial lysates were prepared by disrupting cells suspended in
50 mM Hepes, pH 6.5?1 mM EDTA?5 mM neocuproine, using
Zirconia beads in a Mini Bead Beater (Stratech Scientific,
Luton, U.K.) for 10 s at maximum setting.
Biotin-Switch Assay and Protein Identification. The biotin-switch
assay (16) used anti-biotin mAb from Sigma. Biotin-labeled
proteins were purified by preadsorbing the biotin-HPDP-labeled
lysates onto Sepharose 4B, incubating protein lysates with aga-
rose beads coupled to streptavidin and eluting biotin-HPDP-
labeled proteins with the addition of 2-mercaptoethanol (2-ME)
to a final concentration of 100 mM. Endogenously biotinylated
proteins were purified from bacterial lysates by incubation using
This paper was submitted directly (Track II) to the PNAS office.
intermediate; biotin-HPDP, N-[6-(biotinamido)hexyl]-3?-(2?-pyridyldithio)propionamide;
2-ME, 2-mercaptoethanol; Lpd, lipoamide dehydrogenase; SNAP, S-nitrosoacetylpenicilla-
mine; Mpa, mycobacterial proteasome ATPase; GSNO, S-nitrosoglutathione; KatG, cata-
lase; pca, pyruvate carboxylase; accA3, acetyl CoA carboxylase.
?To whom correspondence should be addressed at: Department of Microbiology and
Immunology, Weill Medical College of Cornell University, 1300 York Avenue, Box 65, New
York, NY 10021. E-mail: email@example.com.
© 2004 by The National Academy of Sciences of the USA
January 11, 2005 ?
vol. 102 ?
no. 2 ?
agarose beads coupled to monomeric avidin (Pierce) and eluted
with free D-biotin at a final concentration of 2 mM. Purified
proteins were separated by SDS?PAGE across an 8% polyacryl-
amide gel. Differentially detected species were excised, digested
with trypsin, batch-fractionated on RP microtips, and the pep-
tide mixtures were analyzed by MALDI-reTOF MS (UltraFlex
TOF?TOF, Bruker Daltonics, Billerica, MA) (20, 21). Selected
protein database (NR; ?1.7 ? 106entries; National Center
for Biotechnology Information, Bethesda), by using the
PEPTIDESEARCH (M. Mann, Center for Eksperimentel Bioinfor-
matik, University of Southern Denmark, Odense, Denmark)
MALDI-TOF?TOF (MS?MS) analysis of the same samples,
using the UltraFlex instrument in ‘‘LIFT’’ mode. Fragment ion
spectra were taken to search the NR by using the MASCOT
MS?MS Ion Search program (Matrix Science, London). A
molecular mass range of up to twice the predicted was covered,
with a mass accuracy restriction ?50 ppm.
Enzyme Assays. Mtb lipoamide dehydrogenase (Lpd) was over-
expressed in Escherichia coli by using its natural coding se-
quence, purified, and assayed as described (22). Purified, re-
combinant Lpd was nitrosylated by incubation with 1 mM
S-nitrosoacetylpenicillamine (SNAP) for 30 min at room tem-
perature in the dark. SNAP and product N-acetylpenicillamine
were removed by serial passage across a MicroBioSpin 6 gel
filtration device (Bio-Rad).
Purified recombinant mycobacterial proteasome ATPase (Mpa)
containing a C-terminal hexahistidine affinity tag was provided as
a generous gift from G. Lin in our laboratory and assayed as
described (23, 24). Purified, hexahistidine-tagged Mpa was nitrosy-
min at room temperature in the dark. Unincorporated GSNO and
product glutathione were removed by serial passage across a
MicroBioSpin 6 gel filtration device (Bio-Rad).
Results and Discussion
We began by recovering intact Mtb from infected bone marrow-
derived primary mouse macrophages to evaluate whether protein
S-nitrosylation occurs in vivo. IFN-?-activated wild-type macro-
phages produced NO (part of which underwent spontaneous oxi-
dation to yield nitrite), and Mtb recovered from these cells con-
tained at least 10 S-nitrosylated proteins. IFN-?-treated NOS2?/?
macrophages, used as a control, produced no nitrite, and Mtb
(Fig. 1a). Thus, Mtb proteins are physiologically S-nitrosylated in
the phagosome of immunologically activated macrophages.
To identify S-nitroso proteins in Mtb systematically, it was
necessary to scale-up, making it impractical to use macrophages
as RNI generators. Choice of an in vitro RNI-generating system
was based on the following considerations. NOS2-derived RNIs
are slowly but potently bactericidal against Mtb in vitro (5).
NOS2-deficient mice fail to control replication of Mtb, succumb-
ing to infection more rapidly than their wild-type counterparts in
acute infections (1, 3). During the chronic phase of infection,
Mtb-infected wild-type mice fed a highly specific NOS2 inhibitor
succumb quickly with a high bacillary burden, whereas counter-
parts fed an inactive enantiomer continue to control the infec-
tion (1). The composition of the milieu in the Mtb-containing
phagosome is unknown. However, the Mtb phagosome of acti-
vated macrophages is acidic (25) and is expected to contain
nitrite, a freely diffusible product of NO autoxidation. Tran-
scriptional adaptation of Mtb within the phagosome of the
activated macrophage depends on macrophage expression of
NOS2, and a large component of that response can be mimicked
by exposing cultures of Mtb to mildly acidified nitrite (26). In
contrast, the phagocyte oxidase is not required for mice to
control Mtb infection (25), in part because of the protection
afforded by Mtb’s katG-encoded KatG peroxidase (27). Finally,
the brief activity of phagocyte oxidase after uptake of Mtb (28)
precedes the induction of NOS2, limiting the amount of per-
oxynitrite that might arise in the phagosome from the mutual
reaction of O2
enzymes. Peroxynitrite often leads to protein tyrosine nitration,
but we could detect no such modification of Mtb proteins in
intraphagosomal Mtb (data not shown). In short, mildly acidified
nitrite is likely to reproduce a major component of the nitrosa-
tive and oxidative environment that Mtb encounters over a
prolonged period in the phagosome of activated macrophages.
Accordingly, suspensions of Mtb were treated with nitrite at pH
5.5. As the acidity of the medium approaches a pH of ?4.5, similar
to that of an Mtb-containing phagosome in an IFN-?-activated
?and NO, the short-lived products of those two
nitrite-treated bacterial cultures. Shown are anti-biotin immunoblots of Mtb
lysates processed by means of the biotin-switch method. Numbers to left are
Mrmarkers. (a) Intracellular Mtb was recovered 16 h postinfection of resting
(?) and IFN-?-stimulated (?) (100 units?ml) bone marrow-derived macro-
phages from wild-type or NOS2-deficient C57BL?6J mice. Arrows denote
(b) S-nitrosylated proteins formed in intact Mtb exposed to nitrite under
conditions that reduced the viable count by a factor of 104without lysing the
bacteria. Arrows denote biotin-HPDP-specific signals indicative of S-
nitrosylated proteins. Stars mark endogenously biotinylated proteins whose
signals increased (filled stars) or decreased (open stars) after exposure to
nitrite. (Right) Confirmation that these signals arose from endogenous rather
than exogenous biotin is shown. Two asterisks, on the far left, indicate
background signals arising from incomplete alkylation (blocking) of cysteine
sulfhydryls. (c) Dose-dependent and RNI-specific origin of anti-biotin signals.
Symbols are as in b.
S-nitrosylation of Mtb proteins in IFN-?-treated macrophages and
www.pnas.org?cgi?doi?10.1073?pnas.0406133102 Rhee et al.
0.5 mM nitrite in 0.5 ml generates about as much NO as 3 ? 105
nitrite required to kill Mtb increases with bacterial culture density
and decreases with time of exposure (30). Thus, to recover suffi-
cient biomass while keeping Mtb intact, we used millimolar con-
centrations of nitrite and short treatment times, then removed
nitrite and any adsorbed proteins with extensive detergent washes
and concentrated bacteria by centrifugation. Exposure of Mtb at
for 12 h at 37°C produced a 4 log10reduction in colony-forming
units without loss of sedimentable organisms. Treatment with an
equimolar concentration of nitrate (pH 5.5), which does not
generate RNIs, did not affect bacterial viability and served as a
control (30, 31).
Mtb treated under these conditions was then physically dis-
rupted and subjected to the biotin-switch assay (Fig. 1b). Omis-
sion of the exogenous biotinylated reagent revealed three en-
dogenously biotinylated species. Peptide mass fingerprinting
confirmed two of these species to contain the biotinylated
subunit of acetyl CoA carboxylase (AccA3) (encoded by the
accA3 gene) and one to contain pyruvate carboxylase (Pca)
(encoded by the pca gene). These biotinylated proteins are
conserved in all organisms (32, 33). With the inclusion of the
biotin label, multiple S-nitrosylated proteins were observed, but
only in lysates prepared from Mtb exposed to nitrite. These
effects were concentration-dependent and no S-nitrosylation
was observed in the presence of an equimolar concentration of
nitrate (Fig. 1c). S-nitrosylated proteins detected in nitrite-
treated cultures of Mtb included species migrating at the same
apparent Mras Mtb proteins S-nitrosylated in the phagosome of
IFN-?-activated macrophages. Unexpectedly, signals from the
endogenously biotinylated proteins of Mtb were also affected by
nitrite in a dose- and RNI-dependent manner, as will be dis-
S-nitrosylated proteins were enriched by streptavidin-based
affinity chromatography, selectively released from their sulfhy-
dryl-derivatized biotin labels with 2-ME and identified by a
combination of peptide mass fingerprinting using MALDI-TOF
MS, and MS sequencing using MALDI-TOF?TOF MS?MS. In
this way, 29 proteins were unambiguously identified (Table 1).
For simplicity, these proteins will be referred to as the S-nitroso
proteome, recognizing limitations inherent to proteomic tech-
niques, such as a bias against trace and insoluble proteins.
The S-nitroso proteome of Mtb proved to be highly enriched for
proteins encoded by genes predicted to be essential or required for
optimal growth based on transposon-mediated insertional mu-
tagenesis (62% of the S-nitroso proteome vs. 15% of the Mtb
predominantly of enzymes involved in intermediary and lipid
metabolism. This functional composition is disproportionate to the
distribution of Mtb genes encoding such functions. For example,
62% of the enzymes in the S-nitroso proteome but only 30% of
Likewise, 14% of the enzymes of the S-nitroso proteome but only
6% of the ORFs in the Mtb genome are annotated to function in
lipid metabolism. The composition of the S-nitroso proteome also
does not correspond to the most abundant proteins of the input
in published 2D gels (55) (data not shown).
Five proteins (19%) of the S-nitroso proteome (phosphoenol-
pyruvate carboxykinase, Mpa, glutamine synthetase, mycocerosic
acid synthase, and acetohydroxyacid synthase) have been individ-
ually validated as important for virulence and?or persistence in
animal models of tuberculosis through targeted gene disruption or
enzymatic inhibitor studies (24, 31, 35–38). Ten proteins (?50% of
are involved in pathways important for the virulence and?or
persistence of Mtb: mycolic acid synthesis (mycocerosic acid syn-
thase, polyketide synthase 13, and fatty acyl-AMP ligase), glucone-
ogenesis (phosphoenolpyruvate carboxykinase and malate syn-
thase), branched chain amino acid synthesis (acetohydroxyacid
synthase), nitrogen assimilation (asparagine synthase, glutamine
synthetase, and glutamate synthase), and iron metabolism (myco-
bacterial ortholog of bacterioferritin) (35–41).
Two enzymes, Lpd and Mpa, were specifically implicated in
the defense of Mtb against peroxynitrite, peroxides, or nitrite
(24, 31, 42). Also identified was catalase (KatG), an enzyme
crucial to Mtb’s defense against reactive oxygen intermediates as
revealed when NOS2 is absent (27). Thus, in addition to enzymes
of intermediary and lipid metabolism, the Mtb S-nitroso pro-
teome includes enzymes that participate in Mtb’s defenses
against oxidative or nitrosative injury.
Three (DnaK, GroEL2, and Tuf) of the 29 proteins (10%) are
devoid of cysteine residues and might be considered false-positives
of the assay (33). However, these proteins were not detected in
eluted from streptavidin columns with the addition of 2-ME. Thus,
these proteins appear to have been captured by complexing with
S-nitrosylated proteins through their chaperonin activities (43–45).
Remarkably, in contrast to their homologs in E. coli, 11 of the 16
(69%) predicted heat shock?chaperonin proteins of Mtb, including
fact may imply a relative resistance of the Mtb chaperone machin-
ery to oxidative stress.
As noted earlier, treatment of Mtb with nitrite also induced
dose-dependent increases in the anti-biotin immunoreactivity of
one endogenously biotinylated species and dose-dependent de-
in lysates prepared from Mtb treated with equimolar concen-
trations of nitrate. With the addition of the thiol-specific reduc-
tant, 2-ME, in the absence or presence of additional nitrite, the
biotinylated species of the highest apparent Mrdisappeared with
an accompanying increase in the signal intensities of the two
smaller biotinylated species. Thus, the largest biotinylated spe-
cies is a disulfide-bonded complex containing a biotinylated
protein. In the presence of nitrite, formation of this complex was
enhanced. These biotinylated species were purified by mono-
meric avidin chromatography and identified by peptide mass
fingerprinting. The two smaller species were identified as the
accA3), which catalyzes the conversion of acetyl CoA to malonyl
CoA, the first committed step in fatty acid biosynthesis, and
pyruvate carboxylase (encoded by pca), which catalyzes the
conversion of pyruvate to oxaloacetate and serves an essential
anaplerotic role during periods of metabolic shunting. The
largest biotinylated species contained both AccA3 and catalase
(encoded by katG), a protein involved in antioxidant defense,
also identified as a target for S-nitrosylation. AccA3 and Pca are
structurally homologous, biotin-dependent, multidomain com-
plexes that contain highly conserved cysteine residues, and
whose assembly is associated with the self-interaction of two
biotin-containing domains (encoded by accA3 and pca, respec-
tively) (46, 47). In solution, KatG similarly exists as a homodimer
and contains a highly conserved cysteine (48). Thus, it is possible
that, within the cell, RNIs can oxidize cysteine thiols to generate
mixed intermolecular disulfide bonds (49). Such mixed disulfide
formation may interfere with the proper assembly and function
or alter the stability of proteins such as KatG, in which isosteric
mutation of a highly conserved cysteine to serine resulted in
increased proteolytic degradation (50).
The techniques used to define the S-nitroso proteome do not
reveal the proportion of each target protein that is S-
nitrosylated. However, because the total quantity of AccA3 and
Pca is represented by their anti-biotin signals, the impact of RNIs
Rhee et al.
January 11, 2005 ?
vol. 102 ?
no. 2 ?
on the endogenously biotinylated proteins of Mtb is revealed as
complete. The nitrite-dependent loss of the anti-biotin signal
from Pca may reflect the formation of a mixed disulfide with Pca
that masks its biotin epitope. Thus, in addition to S-nitrosylation
and methionine oxidation (6), nitrite can give rise to species that
oxidize cysteine thiols, leading to the formation of mixed,
intermolecular, disulfide-bound complexes.
To test the functional impact of S-nitrosylation on protein
function, we selected two proteins for study in vitro, Lpd (encoded
by Rv0462) and Mpa (encoded by Rv2115c). Lpd is the E3 com-
ponent of the pyruvate dehydrogenase complex and also forms an
integral component of Mtb’s peroxynitrite reductase?peroxidase
(42). Transposon-mediated insertional mutagenesis identified lpd
as a gene whose function in Mtb is either essential or required for
normal growth (34). The catalytic activity of Lpd depends on two
highly conserved cysteine residues (22). mpa encodes a mycobac-
terial proteasome-associated ATPase recently identified as a com-
ponent of Mtb’s RNI defense system (24, 31). Mpa contains three
cysteine residues, all of which are conserved in Mycobacterium
leprae, and one that lies close to the ATP-binding site (33).
Purified, recombinant Lpd or Mpa was incubated with a molar
excess of SNAP or GSNO, respectively, and S-nitrosylated, as
confirmed by the biotin-switch method (Fig. 3 a and c). S-
nitrosylation was associated with inhibition of both substrate-
Table 1. Mtb S-nitroso proteome
fragments per protein that covered ?23% of the peptide sequences of the proteins studied, including represen-
tation of both N and C termini of the protein in almost all cases. The observed peptide masses deviated from the
expected masses by an average of 0.02 atomic mass units.
*Genes whose functions are predicted to be essential or required for optimal growth based on transposon-
mediated insertional mutational analysis of Sassetti et al. (40).
www.pnas.org?cgi?doi?10.1073?pnas.0406133102Rhee et al.
dependent, Lpd-mediated consumption of NADH (Fig. 3b) and
Mpa-mediated hydrolysis of ATP (Fig. 3c). S-nitrosylation and
enzymatic inhibition of both Lpd (Fig. 3 a and b) and Mpa (data
not shown) were concentration-dependent and reversed by
treatment with the thiol-specific reductant, 2-ME (data not
shown). There was no inhibition of enzyme activity after incu-
bation of Lpd or Mpa with the nitrosylation-incompetent deriv-
atives (N-acetylpenicillamine or glutathione, respectively) or
vehicle alone (Fig. 3 b and c). Thus, S-nitrosylation inhibits the
enzymatic activities of both Lpd and Mpa.
What is the biophysical basis for the selectivity of the S-nitroso
proteome? NO or nitrosonium ion (NO?) reacts preferentially
with the thiolates of cysteine residues when the pKa of the
sulhydryl is lowered by nearby cationic side chains on other
amino acids (7). Moreover, micellar catalysis favors S-
nitrosylation in hydrophobic environments where NO can accu-
mulate (51). Thus, S-nitrosylation may be favored on cysteine
thiolates in protein interiors. These conditions characterize
cysteines that participate in active-site chemistry. Enzymes that
use active-site cysteines often have redox activity. Together,
these features may explain, in part, why so few proteins were
found to be S-nitrosylated, why so many of the proteins are
enzymes, and why so many of the enzymes are redox-active.
Indeed, of the ?100 S-nitroso proteins identified through in vitro
studies of individual proteins and proteomic surveys of brain,
mesangium, endothelium, and mitochondria, nearly 60% repre-
sent enzymes, among which are the mammalian orthologs of Lpd
and aconitase (7, 52–54). The remainder of described S-nitroso
proteins are largely proteins involved in channels, transport,
structure, DNA binding, and storage, including the mammalian
ortholog of bacterioferritin (7).
RNIs are broadly active as antimicrobial molecules, but can-
didate targets have not been identified systematically. The
remarkable convergence of the Mtb S-nitroso proteome with
known virulence and?or persistence factors important to the
pathogenesis of tuberculosis supports a significant role for
protein S-nitrosylation as an effector of the antimicrobial activ-
ities of RNIs. The relative contribution of S-nitrosylation to the
antimicrobial activities of RNIs awaits detailed studies of indi-
vidual target proteins to evaluate the specificity and stoichiom-
etry of S-nitrosylation-dependent effects. That RNIs can target
three proteins important in the antioxidant defense system of
Mtb (Lpd, Mpa, and KatG) emphasizes the dynamic interplay
between the pathogen’s efforts to subvert host immune re-
sponses and the host’s efforts to disable these same microbial
targets inhibited in Mtb by the host immune system may point to
useful targets for the development of antitubercular drugs.
We thank G. Lin for purified, recombinant Mpa; R. Bryk for expert
advice; and W. D. Johnson, Jr., for enthusiastic support of this work. This
work was supported by National Institutes of Health Grants P01AI56293
(to C.F.N.) and T32AI007613 and K08AI0061393 (to K.Y.R.). The
Department of Microbiology and Immunology is supported by the
William Randolph Hearst Foundation.
1. MacMicking, J. D., North, R. J., LaCourse, R., Mudgett, J. S., Shah, S. K. &
Nathan, C. F. (1997) Proc. Natl. Acad. Sci. USA 94, 5243–5248.
2. Flynn, J. L., Scanga, C. A., Tanaka, K. E. & Chan, J. (1998) J. Immunol. 160,
3. Scanga, C. A., Mohan, V. P., Tanaka, K., Alland, D., Flynn, J. L. & Chan, J.
(2001) Infect. Immun. 69, 7711–7717.
4. Choi, H. S., Rai, P. R., Chu, H. W., Cool, C. & Chan, E. D. (2002) Am. J. Respir.
Crit. Care Med. 166, 178–186.
onstrates nitrite-induced, thiol-sensitive complex formation of endogenously
biotinylated Mtb proteins. The anti-biotin signal ? was identified by peptide
mass fingerprinting as the biotin carboxylase-biotin carboxyl carrier protein
subunit of AccA3 and ? as Pca. The species labeled ? consisted of both AccA3
RNI-induced intermolecular disulfide formation involving AccA3 and
dehydrogenase (Lpd; Rv0462) and mycobacterial proteasome-associated AT-
Pase (Mpa; Rv2115c). (a Left Upper) Anti-biotin immunoblotting of purified,
recombinant Mtb Lpd incubated with SNAP, N-acetylpenicllamine (AP), or
vehicle (H2O) and assayed by using the biotin-switch method. (Right Upper)
Anti-biotin immunoblotting of purified, recombinant Lpd incubated with
vehicle, 2 mM SNAP, or 2 mM SNAP, followed by treatment with the thiol-
presented were taken from a single gel with intervening lanes removed.)
(Lower) Ponceau S staining of nitrocellulose membrane before anti-biotin
immunoblotting. (b) Effects of S-nitrosylation on Lpd-catalyzed lipoamide-
dependent NADH consumption in the presence of a saturating concentration
of substrate under initial rate conditions. Data are means ? SD for four
independent experiments. (c Left) Anti-biotin immunoblotting of purified,
recombinant Mtb Mpa incubated with GSNO, glutathione (GSH), or vehicle
(H2O) and assayed by using the biotin-switch method. (Right) Effects of
S-nitrosylation on Mpa-catalyzed ATP hydrolysis in the presence of a saturat-
ing concentration of substrate under initial rate conditions. Means ? SD for
three independent experiments are shown.
S-nitrosylation is associated with inhibition of dihydrolipoamide
Rhee et al.
January 11, 2005 ?
vol. 102 ?
no. 2 ?
5. Nathan, C. F. & Ehrt, S. (2004) in Tuberculosis, eds. Rom, W. N. & Garay, S. M. Download full-text
(Lippincott, Williams & Wilkins, Philadelphia), 2nd. Ed., pp. 215–235.
6. St. John, G., Brot, N., Ruan, J., Erdjument-Bromage, H., Tempst, P., Weiss-
bach, H. & Nathan, C. (2001) Proc. Natl. Acad. Sci. USA 98, 9901–9906.
7. Stamler, J. S., Lamas, S. & Fang, F. C. (2001) Cell 106, 675–683.
8. Imlay, J. A. (2003) Annu. Rev. Microbiol. 57, 395–418.
9. Salvati, L., Mattu, M., Colasanti, M., Scalone, A., Venturini, G., Gradoni, L.
& Ascenzi, P. (2001) Biochim. Biophys. Acta 1545, 357–366.
10. Saura, M., Zaragoza, C., McMillan, A., Quick, R. A., Hohenadl, C., Lowen-
stein, J. M. & Lowenstein, C. J. (1999) Immunity 10, 21–28.
11. Persichini, T., Colasanti, M., Lauro, G. M. & Ascenzi, P. (1998) Biochem.
Biophys. Res. Commun. 250, 575–576.
12. Schapiro, J. M., Libby, S. J. & Fang, F. C. (2003) Proc. Natl. Acad. Sci. USA
13. Venturini, G., Salvati, L., Muolo, M., Colasanti, M., Gradoni, L. & Ascenzi, P.
(2000) Biochem. Biophys. Res. Commun. 270, 437–441.
14. Venturini, G., Colasanti, M., Salvati, L., Gradoni, L. & Ascenzi, P. (2000)
Biochem. Biophys. Res. Commun. 267, 190–193.
15. Nathan, C. & Shiloh, M. U. (2000) Proc. Natl. Acad. Sci. USA 97, 8841–8848.
16. Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. & Snyder,
S. H. (2001) Nat. Cell Biol. 3, 193–197.
17. Parish, T. (2003) J. Bacteriol. 185, 6702–6706.
18. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. (2002)
Mol. Microbiol. 43, 717–731.
19. Ding, A. H. & Nathan, C. F. (1987) J. Immunol. 139, 1971–1977.
20. Winkler, G. S., Lacomis, L., Philip, J., Erdjument-Bromage, H., Svejstrup, J. Q.
& Tempst, P. (2002) Methods 26, 260–269.
21. Erdjument-Bromage, H., Lui, M., Lacomis, L., Grewal, A., Annan, R. S.,
McNulty, D. E., Carr, S. A. & Tempst, P. (1998) J. Chromatogr. A 826, 167–181.
22. Argyrou, A. & Blanchard, J. S. (2001) Biochemistry 40, 11353–11363.
23. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. & Candia, O. A. (1979) Anal.
Biochem. 100, 95–97.
24. Darwin, K. H., Lin, G., Chen, Z., Li, H. & Nathan, C. (2004) Mol. Microbiol.,
25. MacMicking, J. D., Taylor, G. A. & McKinney, J. D. (2003) Science 302,
26. Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan,
I. M., Dolganov, G., Efron, B., Butcher, P. D., Nathan, C. & Schoolnik, G. K.
(2003) J. Exp. Med. 198, 693–704.
27. Ng, V. H., Cox, J. S., Sousa, A. O., MacMicking, J. D. & McKinney, J. D. (2004)
Mol. Microbiol. 52, 1291–1302.
28. Piddington, D. L., Fang, F. C., Laessig, T., Cooper, A. M., Orme, I. M. &
Buchmeier, N. A. (2001) Infect. Immun. 69, 4980–4987.
29. Ehrt, S., Schnappinger, D., Bekiranov, S., Drenkow, J., Shi, S., Gingeras, T. R.,
Gaasterland, T., Schoolnik, G. & Nathan, C. (2001) J. Exp. Med. 194,
30. Ehrt, S., Shiloh, M. U., Ruan, J., Choi, M., Gunzburg, S., Nathan, C., Xie, Q.
& Riley, L. W. (1997) J. Exp. Med. 186, 1885–1896.
31. Darwin, K. H., Ehrt, S., Gutierrez-Ramos, J. C., Weich, N. & Nathan, C. F.
(2003) Science 302, 1963–1966.
H. G. (1988) J. Biol. Chem. 263, 6461–6464.
33. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D.,
Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, et al. (1998) Nature 393,
34. Sassetti, C. M., Boyd, D. H. & Rubin, E. J. (2001) Proc. Natl. Acad. Sci. USA
35. Rousseau, C., Sirakova, T. D., Dubey, V. S., Bordat, Y., Kolattukudy, P. E.,
Gicquel, B. & Jackson, M. (2003) Microbiology 149, 1837–1847.
36. Tullius, M. V., Harth, G. & Horwitz, M. A. (2003) Infect. Immun. 71,
37. Grandoni, J. A., Marta, P. T. & Schloss, J. V. (1998) J. Antimicrob. Chemother.
38. Liu, K., Yu, J. & Russell, D. G. (2003) Microbiology 149, 1829–1835.
39. Portevin, D., De Sousa-D’Auria, C., Houssin, C., Grimaldi, C., Chami, M.,
Daffe, M. & Guilhot, C. (2004) Proc. Natl. Acad. Sci. USA 101, 314–319.
40. Sassetti, C. M., Boyd, D. H. & Rubin, E. J. (2003) Mol. Microbiol. 48, 77–84.
41. Timm, J., Post, F. A., Bekker, L. G., Walther, G. B., Wainwright, H. C.,
Manganelli, R., Chan, W. T., Tsenova, L., Gold, B., Smith, I., et al. (2003) Proc.
Natl. Acad. Sci. USA 100, 14321–14326.
Science 295, 1073–1077.
43. Gilbert, H. F. (1984) Methods Enzymol. 107, 330–351.
44. Smith, K. E., Voziyan, P. A. & Fisher, M. T. (1998) J. Biol. Chem. 273,
45. Echave, P., Esparza-Ceron, M. A., Cabiscol, E., Tamarit, J., Ros, J., Membrillo-
Hernandez, J. & Lin, E. C. (2002) Proc. Natl. Acad. Sci. USA 99, 4626–4631.
46. Choi-Rhee, E. & Cronan, J. E. (2003) J. Biol. Chem. 278, 30806–30812.
47. Sueda, S., Islam, M. N. & Kondo, H. (2004) Eur. J. Biochem. 271, 1391–1400.
48. Nagy, J. M., Cass, A. E. & Brown, K. A. (1997) J. Biol. Chem. 272, 31265–31271.
49. Jourd’heuil, D., Jourd’heuil, F. L. & Feelisch, M. (2003) J. Biol. Chem. 278,
50. Saint-Joanis, B., Souchon, H., Wilming, M., Johnsson, K., Alzari, P. M. & Cole,
S. T. (1999) Biochem. J. 338, 753–760.
51. Nedospasov, A., Rafikov, R., Beda, N. & Nudler, E. (2000) Proc. Natl. Acad.
Sci. USA 97, 13543–13548.
52. Kuncewicz, T., Sheta, E. A., Goldknopf, I. L. & Kone, B. C. (2003) Mol. Cell.
Proteomics 2, 156–163.
53. Foster, M. W. & Stamler, J. S. (2004) J. Biol. Chem. 279, 25891–25897.
54. Martinez-Ruiz, A. & Lamas, S. (2004) Arch. Biochem. Biophys. 423, 192–199.
55. Pleissner, K. F., Eifert, T., Buettner, S., Schmidt, F., Boehme, M., Meyer, T. F.,
Kaufmann, S. H. & Jungblut, P. R. (2004) Proteomics 4, 1305–1313.
www.pnas.org?cgi?doi?10.1073?pnas.0406133102 Rhee et al.