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J. Biol. Chem., Vol. 277, Issue 15, 12824-12829, April 12, 2002
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From the Department of Pharmaceutical Chemistry, University of
California, San Francisco, California 94143-0446
Received for publication, November 8, 2001, and in revised form, January 25, 2002
Ethionamide (ETA), a prodrug that must undergo
metabolic activation to exert its cytotoxic effects, is a second line
drug against tuberculosis, a disease that infects more than a third of
the world's population. It has been proposed, on the basis of genetic
experiments, that ETA is activated in Mycobacterium tuberculosis by the protein encoded by the gene Rv3854c
(DeBarber, A. E., Mdluli, K., Bosman, M., Bekker, L.-G.,
and Barry, C. E., III (2000) Proc. Natl. Acad. Sci.
U. S. A. 97, 9677-9682; Baulard, A. R., Betts, J. C., Engohang-Ndong, J., Quan, S., McAdam, R. A., Brennan, P. J.,
Locht, C., and Besra, G. S. (2000) J. Biol. Chem.
275, 28326-28331). We report here the expression, purification, and
characterization of the protein encoded by this gene. Our results
establish that the enzyme (EtaA) is an FAD-containing enzyme that
oxidizes ETA to the corresponding S-oxide. The
S-oxide, which has a similar biological activity as ETA, is
further oxidized by EtaA to 2-ethyl-4-amidopyridine, presumably via the
unstable doubly oxidized sulfinic acid intermediate. This flavoenzyme
also oxidizes thiacetazone, thiobenzamide, and isothionicotinamide and
thus is probably responsible, as suggested by the observation of
crossover resistance, for the oxidative activation of other thioamide
antitubercular drugs.
Tuberculosis continues to be a major worldwide epidemic with
approximately one-third of the world population infected with Mycobacterium tuberculosis, 7 million people each year
developing the active disease, and 2 million deaths per annum (1, 2). Drugs such as isoniazid
(INH)1 and rifampicin have
historically been successful in the treatment of tuberculosis
infections. In recent history, however, poor compliance with the
prolonged and complicated chemotherapeutic regimens currently used to
treat the disease (3), in conjunction with the advent of the AIDS
epidemic and the increased mobility of human populations, has led to
the emergence of numerous multidrug-resistant M. tuberculosis strains (4). Resistance to frontline therapeutics,
most notably INH and rifampicin, results in treatment of patients with
"second-line" agents that are less effective and/or more toxic.
Among these second tier drugs for the treatment of multidrug-resistant
tuberculosis, one of the most effective is ethionamide (ETA) (5).
ETA (1, Fig. 1), like INH, is thought to be a prodrug that
must be converted to its active form by the bacterial cell. Both ETA
and INH, when activated, appear to disrupt cell wall biosynthesis and
have at least one common cellular target, the enoyl-acyl carrier
protein reductase InhA (6, 7). Support for a common site of action can
be deduced from gene array studies demonstrating that both ETA and INH
induce similar patterns of gene expression in M. tuberculosis (8). Despite this evidence for a common site of
action, INH and ETA are activated by different mechanisms as resistance
to INH does not confer resistance to ETA (9). INH is now known to be
oxidized by the bacterial catalase-peroxidase KatG to a reactive
species, probably an acyl free radical, that is eventually responsible
for its bacterial toxicity (10-13). Mutations in KatG that diminish or
annihilate its activity toward INH are responsible for a large
proportion of the INH-resistant M. tuberculosis strains.
Until recently, however, the enzyme that activates ETA has remained
obscure (14, 15). Sequence homology studies using the Rv3854c gene as a
template indicate that the protein responsible for ETA activation may
be a flavin monooxygenase (14, 15). In vivo mammalian and
bacterial studies suggest that the first metabolite of ETA is the
thioamide S-oxide (ETA-SO, 2, Fig. 1), which
retains the biological activity of the parent drug (16-19). Other
metabolites identified in whole cell bacterial systems include the
nitrile (3), amide (4), and alcohol (6) derivatives of ETA (Fig.
1) (14).
Recently two laboratories independently reported identification of a
gene, Rv3854c, in the M. tuberculosis genome that codes for
a protein that activates ETA (14, 15). Genetic and transfection experiments provided strong evidence for the biological role of the
Rv3854c encoded protein (EtaA) in the activation of ETA. However, the
actual enzyme was not isolated, and its nature remains speculative. Herein we report cloning, heterologous expression, purification, and
characterization of the Rv3854c gene product. Our results establish
that the enzyme responsible for ETA activation is an FAD-containing
enzyme, provide information on the catalytic and physical properties of
this enzyme, and demonstrate that it catalyzes two steps rather than
one step in the activation of ETA.
Materials
All chemicals, including NADPH, were purchased from Aldrich or
Sigma, were of ACS grade or better, and were used without further purification. All the enzymes used in cloning procedures were from New
England Biolabs (Beverly, MA) and were used with buffers from the same
company. Protein purification was done at 4 °C. Once purified, the
protein was stored at Instrumentation
UV-visible spectra were obtained on either a Hewlett Packard
model 8254A diode array instrument or a Varian Cary 1E
spectrophotometer. NMR spectra were collected on a Varian Unity 400 MHz
instrument. Mass spectra were obtained using a Thermoquest Finnigan
LCQDECA electrospray ionization (ESI) ion trap spectrometer. HPLC was performed on a Hewlett Packard Series 1090 liquid chromatograph equipped with either a Rheodyne model 7125 manual sample injector or an autosampler.
Kinetic Assays
All activity and steady state kinetic assays were conducted by
monitoring the formation of the product ETA-SO (2). Product
formation was measured at 350 nm over a time of 5-10 min using the
Cary 1E split beam spectrophotometer. The molar extinction coefficient
of ETA-SO in the reaction system was experimentally determined to be
6,000 (±4%) M The enzymatic reaction was typically initiated by adding substrate
(ETA) to the sample cuvette containing the reaction medium plus
the enzyme and an equivalent volume of acetonitrile to the reference
cuvette. The reference cell contained all solution components except
for enzyme and substrate (ETA), allowing for accurate correction of any
NADPH absorption in the region of interest. Prior to addition of
substrate, the reaction and reference mixtures were thermally equilibrated in the cuvettes at 37 °C for 2 min.
Synthesis of Proposed Metabolites
ETA-SO (2)--
ETA-SO was prepared from ETA
following a modification of a published protocol (20). ETA (1.45 g,
8.72 mmol) was suspended in absolute ethanol (7 ml) immersed in a room
temperature water bath. Hydrogen peroxide (30% (v/v), 922 µl, 9.59 mmol) was added slowly while monitoring the solution temperature. The
temperature was never allowed to exceed 35 °C. After all of the
H2O2 was added, the solution was stirred at
room temperature for 45 min. The reaction was monitored by TLC (silica
on plastic, eluent: 90% ethyl acetate, 10% methanol) and quenched
with 3 ml of water when the reaction was ~90% complete. The solvent
was removed in vacuo resulting in an orange oil.
Recrystallization at 2-Ethyl-4-cyanopyridine (3)--
The reaction was carried
out under a dry argon atmosphere with flame-dried glassware. Following
a published procedure (21), CH2Cl2 (10 ml) was
transferred by syringe to a round bottom flask charged with ETA (333.5 mg, 2.01 mmol), and the suspension was cooled to 0 °C. First
triethylamine (635 µl, 4.50 mmol) and then 2,2,2-trichloroethyl
chloroformate (315 µl, 2.29 mmol) were added dropwise. The reaction
was monitored by silica TLC (eluent: 30% ethyl acetate in hexanes) for
product formation. The solution was stirred under argon for 1.5 h,
allowing it to warm to room temperature. The reaction was quenched with
water (10 ml) and extracted with CH2Cl2 (2 × 15 ml). The organic layer was then washed with brine and dried with
Na2SO4. The solvent was then removed in
vacuo, and the resultant residue was purified on a silica column
(12.5 × 2.3 cm, l × diameter) packed in a mixture of
5% ethyl acetate in hexanes. The product was eluted using a gradient
of 10-15% ethyl acetate in hexanes (5%/100 ml). This procedure
yielded 192 mg (1.45 mmol) of pure product (72% purified yield) as a
pale yellow oil: IR (neat) 2238 cm 2-Ethyl-4-amidopyridine (4)--
Following a
published procedure (22), the amide was synthesized from the nitrile
under phase transfer catalytic conditions. A solution of 3 (84 mg, 0.635 mmol) and tetrabutylammonium tetrafluoroborate (62 mg,
0.188 mmol) in CH2Cl2 (0.318 ml) was cooled to
0 °C in an ice bath. To the cooled solution, 30% aqueous H2O2 (0.424 ml) was slowly added followed by
20% (w/w) aqueous NaOH (0.339 ml). The solution was vigorously stirred
for 2 h, allowing the solution to warm to room temperature. The
reaction mixture was extracted with CH2Cl2 (10 ml), and the organic layer washed with an equal volume of brine and
dried over Na2SO4. The solvent was removed
in vacuo, and the residue was purified on an alumina column
(8.5 × 2.3 cm, l × diameter) packed in ethyl acetate. The
product was eluted using a gradient of 0-20%
CH2Cl2 in ethyl acetate (5%/100 ml). This
procedure yielded 57 mg (0.38 mmol) of pure product (50% purified
yield) as a white solid: 1H NMR
(d6-Me2SO, 400 MHz) [ 2-Ethyl-4-carboxypyridine (5)--
The nitrile,
3 (529 mg, 4 mmol), was dissolved in water (10 ml).
Concentrated H2SO4 (4 eq) was then added, and
the solution was refluxed for 4 h. The reaction mixture was
extracted with CH2Cl2 to remove the residual
starting material before the aqueous layer was basified to pH = 10 with aqueous NaOH. The water was evaporated in vacuo, and
the residue was redissolved in methanol and filtered. The sodium salt
of the acid (153 mg, 0.884 mmol) was obtained as a yellowish powder
following evaporation of the methanol: 1H NMR
(CD3OD, 400 MHz) [ Thiobenzamide S-Oxide (8)--
Thiobenzamide (686 mg, 5 mmol) was dissolved in pyridine (5 ml), and the solution was
warmed to 30 °C before H2O2 (30%, 1.2 eq)
was added dropwise. The solution was stirred for 2 h at room temperature, and the pyridine was then removed under reduced pressure to yield yellow crystals of thiobenzamide S-oxide: TLC
analysis (5% ethyl acetate, pentane) RF
(starting material) = 0.22, RF (product) = 0.61; 1H NMR (CDCl3, 400 MHz) [ Protein Expression and Purification
The gene was obtained as an insert in the pMH29 vector from
Clifton Barry III and Andrea E. DeBarber (National Institutes of
Health). PCR was used to amplify the insert with NdeI and
HindIII restriction sites at the 5' and 3' termini,
respectively. The primers used for the PCR were: 5'-end,
5'-GCGTGGACATATGACCGAGCACCTCG; 3'-end,
5'-CTAAAGCTTCGCTAAAGCTAAACCCCC (the restriction sites are
underlined). The insert was ligated into the pCWori expression vector
containing a poly-His insert at the 5'-end of the coding sequence just
prior to the start codon. Sequencing was used to confirm that the
full-length gene without alterations in the sequence was inserted into
the expression vector. The expression vector was transformed into
commercial (Invitrogen) competent DH5 The E. coli cells were resuspended in 50 mM Tris
(300 mM NaCl, 1% (v/v) Triton X-100, 6 mM
imidazole, pH = 7.4), lysed, and sonicated. The cytosolic fraction
was removed after centrifugation at 25,000 × g. This
supernatant was loaded directly onto a Ni-NTA affinity column
pre-equilibrated with 50 mM Tris, 500 mM NaCl, 10% glycerol, pH = 7.4. After loading, the column was washed with 10 column volumes of the same buffer, and the protein was eluted with a
gradient over 10 column volumes of 0-200 mM imidazole in 50 mM Tris (pH = 7.4) containing 500 mM
NaCl and 10% glycerol. The fractions containing the pure Rv3854c gene
product (EtaA), as determined by sodium dodecylsulfate polyacrylamide
gel electrophoresis, were pooled and concentrated. Imidazole was
removed from the protein solution by passing it through a Sephadex
G-25M size exclusion column (elution buffer: 50 mM Tris,
100 mM KCl, 10% glycerol, pH = 7.4) with a final
yield of ~15 mg of pure protein/liter of culture.
Fast protein liquid chromatographic analysis of the protein was done on
a Superdex 75 FPLC column eluted at a rate of 0.5 ml/min with 50 mM Tris buffer, pH = 7.4, containing 100 mM KCl and 10% glycerol. Thin layer chromatographic
comparison of the prosthetic group extracted from the recombinant
flavoprotein with authentic FMN and FAD was done on silica gel using
water as the elution solvent. The flavins could be detected visually
due to their bright yellow/orange color.
HPLC Identification of Metabolites
The enzymatic reactions with ETA, thiobenzamide,
isothionicotinamide, and thiacetazone were analyzed by HPLC, and the
metabolites were identified, where possible, by comparison with
authentic standards. The enzymatic reaction was run for 3 h at
37 °C using conditions identical to those described for the kinetic
assays. The crude reaction mixture was filtered through a 0.22-µm
syringe filter (Millex®-GV low protein binding Durapore
membrane) and analyzed, without further modification, by HPLC on a
Whatman Partisil® C8 reverse-phase column (particle size,
5 µm; 250- × 4.6-mm inner diameter) equipped with a C8 guard column
(particle size, 5 µm; 7.5- × 4.6-mm inner diameter). The solvent
system consisted of 0.01% formic acid in water (A) and 0.01%
formic acid in acetonitrile (B). The mixture was eluted with a gradient
of 1-8.3% B over 30 min. The eluent was monitored at 284 and 350 nm,
and complete UV-visible spectra of the metabolite peaks were collected
for comparison with authentic standards.
Cloning and Expression of the Protein--
Genetic experiments,
including expression in intact cells, have implicated the protein
encoded by Rv3854c in the bioactivation of ETA to a form of the drug
with direct antimycobacterial activity (14, 15). The metabolic products
of ETA suggest that the protein in question is likely to be a
monooxygenase (15). The transformations are consistent with the
involvement of either a cytochrome P450 or a flavin monooxygenase, but
the protein sequence encoded by Rv3854c is more consistent with a
flavoprotein (14, 15). To elucidate the precise nature of the enzyme
involved in the activation of ETA and the nature of the
transformation(s) that it catalyzes, we have cloned and expressed
Rv3854c in E. coli. The protein was expressed with a
poly-His tag to facilitate purification of the protein. Expression of
two constructs, one with the poly-His tag at the amino terminus and the
other with the tag at the carboxyl terminus, gives protein with similar
catalytic properties, indicating that the poly-His tag does not
interfere with catalytic function (all subsequent experiments were
conducted with the amino-terminally His-tagged protein). An exploration
of expression conditions led to production of the enzyme in yields of
15 mg of purified protein/liter of medium. The protein was
highly purified by Ni-NTA affinity chromatography as shown by the fact
that even at high concentrations the
protein gives rise to a single band on SDS-PAGE (Fig. 2). The specific
activity of the protein product at different stages of the purification
procedure, measured as indicated under "Experimental Procedures"
and as discussed below, is shown in Table I. Herein the recombinant
Rv3854c gene product is denoted as
EtaA.
Physical Characterization of the Protein--
EtaA, as expected
from the predicted length of 488 amino acids, migrates with a molecular
mass of ~55 kDa (Fig. 2). FPLC analysis of EtaA on a Superdex column
shows that the protein elutes as a single peak but at a molecular
weight that corresponds to that of an oligomeric (3-4-mer) species,
suggesting that EtaA aggregates in solution. UV-visible spectra of
solutions of EtaA exhibit maxima at 365 and 440 nm in agreement with
its identification as a flavoprotein (Fig.
3). Furthermore, extraction of the
prosthetic group by boiling EtaA for 5 min and sedimenting the
denatured protein by centrifugation yields a supernatant with a
spectrum identical to that of authentic FAD (Fig. 3). Quantitation of
the flavin released from denatured EtaA yields a ratio of flavin to
protein of 1:1.3, indicating that the protein binds one flavin group.
Thin layer chromatographic comparison of the extracted prosthetic group
(RF = 0.9) with authentic FAD (RF = 0.9) and FMN (RF = 0.1) showed that the prosthetic
group has the retention time of FAD and not FMN. Rv3854c clearly codes
for a flavoprotein with a single FAD prosthetic group.
Catalytic Oxidation of ETA by EtaA--
The identity of the
metabolite(s) produced in the reaction of purified EtaA with ETA and
NADPH was determined by letting the reaction proceed for 3 h,
separating the products by reverse-phase HPLC, and comparing the peaks
with authentic (synthetic) standards. The standards that were available
were the S-oxide (2), 2-ethyl-4-cyanopyridine
(3), 2-ethyl-4-amidopyridine (4), and
2-ethyl-4-carboxypyridine (5), all of which have been reported as metabolites from the in vivo mycobacterial
oxidation of ETA (14, 16). The two major metabolic products observed with this method had retention times of 9.1 and 14.9 min. The two peaks
were identified as the amide (4) and ETA-SO (2), respectively, by comparison with the elution time and spectrum of the
synthetic standards (tR(4) = 9.1 and
tR(2) = 14.9 min) (Fig.
4).
Since ETA-SO exhibits similar antimycobacterial activity as ETA (14),
it was important to determine whether the amide metabolite (4) is a product of EtaA metabolism of ETA-SO or a product of an alternate enzymatic oxidation of ETA. The synthetic ETA-SO standard was therefore incubated with EtaA and NADPH as described above
for incubations with ETA. A further oxidative transformation was
detected by HPLC (Fig. 4). The second oxidation product co-elutes with
the 9.1-min metabolite observed in the HPLC analyses of the ETA
incubations, indicating that the enzyme is capable of metabolizing ETA-SO to the amide (4) (Fig. 4).
EtaA was further implicated in the direct metabolism of ETA and ETA-SO
by analysis of 3-h (37 °C) incubations of either ETA or ETA-SO with
and without enzyme in the presence of NADPH and with and without NADPH
in the presence of the enzyme. In both sets of experiments no
metabolism or decomposition of either ETA or ETA-SO was observed by
HPLC when either the enzyme or NADPH was omitted. As might be expected
for a flavoprotein, both NADPH and O2 are required for
formation of both the ETA sulfoxidation product (2) and the
amide (4) by EtaA. Omission of NADPH, or running the
reaction under anaerobic conditions, suppressed product formation.
Furthermore, NADH could not be substituted for NADPH in the reaction
(data not shown).
The catalytic activity of purified EtaA with respect to ETA was
evaluated by measuring the initial rate of ETA-SO formation. The rate
of ETA sulfoxidation was monitored at 350 nm (see "Experimental Procedures" for specific assay conditions). The
Km, Vmax, and
kcat values for the oxidation of ETA were found,
from analysis of Lineweaver-Burke double-reciprocal plots, to be 194 (±3%) µM, 1.46 (±3%) nmol of ETA-SO
min Substrate Specificity--
To determine the degree of substrate
specificity of EtaA, we have examined the oxidation of the ETA
analogues thiobenzamide (7) and isothionicotinamide
(9) (Fig. 6). HPLC analyses of
EtaA incubations with thiobenzamide and isothionicotinamide reveal the
formation in each instance of two observable metabolites (not shown).
The products of the metabolism of thiobenzamide by EtaA have been
confirmed to be the corresponding S-oxide (8) and
benzamide by comparison of their absorption spectra and retention times
with those of synthetic S-oxide and benzamide standards. One
metabolite of the isothionicotinamide reaction was identified as the
amide by comparison with commercially available material. The second
metabolite was tentatively identified as the S-oxide, but an
authentic standard was not available to confirm this assignment. It is
nevertheless clear that both compounds are substrates for EtaA and
undergo an initial oxidation similar to that observed with ETA.
Cloning experiments in M. tuberculosis,
Mycobacterium smegmatis, and Mycobacterium bovis
BCG have shown that Rv3854c codes for a protein that is critical
for the antitubercular activity of ETA (14, 15). These studies also
demonstrated that the expression level of Rv3854c is regulated by a
suppressor gene, Rv3855, and that resistance to ETA increases with
increased expression levels of Rv3855. Conversely increases in the
expression levels of Rv3854c confer heightened sensitivity of the
bacilli to ETA (14, 15). The work presented here demonstrates that the
protein encoded by Rv3854c, EtaA, is a flavoprotein monooxygenase with a single FAD prosthetic group. The identity of EtaA as a flavoprotein, a classification suggested by the presence of a consensus
NX5DX3GXGXXG flavin binding domain in the predicted protein sequence (23), is
clearly established by the demonstration that the protein prosthetic group is a molecule of FAD. The UV-visible spectrum of the pure protein
obtained after a final size-exclusion chromatographic step (Fig. 3) is
typical of a flavoprotein. The Rv3854c protein is most likely
membrane-associated when expressed in the E. coli system as
evidenced by its presence predominantly in the pellet fraction of the
first centrifugation step following cell lysis. The need for
resolubilization of precipitated or membrane-associated protein can be
avoided by introducing Triton X-100 detergent in the lysis buffer. A
poly-His tag has been engineered into the amino terminus of the
protein, allowing for efficient purification to homogeneity via Ni-NTA
affinity chromatography. The expression and purification procedures
delineated in this report allow for the isolation of highly purified,
stable, and active recombinant EtaA that can be utilized in kinetic
and metabolic studies.
The first step in the bioactivation of ETA is an NADPH- and
O2-dependent reaction that yields the
S-oxide metabolite ETA-SO. The Km,
Vmax, and kcat for the
initial oxidation of ETA by purified EtaA indicate that it is a
reasonably good substrate for the enzyme. However, ETA-SO is not so
reactive that it cannot be synthesized, stored, and physically
characterized. These properties of ETA-SO, along with the observation
that it is as lethal to the tubercule bacilli as ETA, suggest that
ETA-SO itself requires further activation to a final cytotoxic species
(15). Incubation of authentic ETA-SO with EtaA under turnover
conditions shows, indeed, that it is further metabolized by the enzyme
to another metabolite. Chromatographic comparison of this second
metabolite with synthesized standards identify it as
2-ethyl-4-amidopyridine (Fig. 4). This second metabolite is thus a
product of the enzymatic action of EtaA on ETA-SO and not a branching
product stemming from an alternative reaction with ETA. However,
4 is likely not the actual enzymatic product of EtaA and
ETA-SO but rather is a more stable molecule resulting from
decomposition of this putative cytotoxic metabolite, possibly a
sulfinic acid species (Fig. 1, 2a). Since the final
metabolite, 4, has no antitubercular activity, the key
species is the reactive intermediate formed by EtaA that is the
precursor of the amide.
Whole cell incubations of M. tuberculosis with
radiolabeled ETA have indicated that ETA-SO and the nitrile
(3) are initially formed and accumulate to a maximum
concentration over a couple of hours (14). Following this early
increase of ETA-SO and 3, their cellular concentrations
begin to decrease, and increases in the amide (4) and
alcohol (6) are observed (14). However, the final
metabolites produced in the intact bacillus may arise both by
alternative metabolic pathways acting on ETA and secondary metabolism
of the products generated by EtaA. The link between the ETA-resistant
tuberculosis and mutations in the Rv3855 and Rv3854c genes (14, 15)
clearly indicates that the activation step is catalyzed by EtaA and
therefore that the oxidation of ETA-SO catalyzed by this enzyme is
critical for activation of the drug to its cytotoxic metabolite.
The natural substrate for the M. tuberculosis flavoprotein
monooxygenase, EtaA, is not known. As bacteria containing mutated, presumably inactive, forms of EtaA are viable (14), the enzyme cannot
be involved in the processing of an essential endogenous substrate. In
agreement with the fact that patients infected with ETA-resistant
M. tuberculosis strains present cross-resistance to
thiacetazone (14), we have shown here that thiobenzamide (7)
and isothionicotinamide (9) are also substrates for EtaA.
Thiobenzamide is oxidized by EtaA to thiobenzamide S-oxide (8) and benzamide. The oxidation of isothionicotinamide (9) by EtaA generates two products, a minor product
unambiguously identified as isonicotinamide by comparison with an
authentic sample and a major product tentatively identified as the
sulfoxide from its HPLC properties. Thus, EtaA, like the mammalian
flavoprotein monooxygenases, exhibits a relatively broad substrate
specificity with the primary constraint being that an appropriate
oxidizable functionality be present.
Thiobenzamide, a substrate for the M. tuberculosis
flavoprotein monooxygenase (see above), is one of a variety of related compounds that are known to be substrates for the mammalian
flavoprotein monooxygenases (24-27). As shown here for the reaction
with the mycobacterial enzyme, thiobenzamide is also oxidized to the
corresponding S-oxide by the mammalian flavoprotein
monooxygenases (17, 18). The thiobenzamide S-oxide is
hepatotoxic in rats (24, 28), but the available evidence suggests that
this hepatotoxicity requires further transformation of the
S-oxide to a secondary reactive metabolite. Thus, rat liver
microsomes oxidize thiobenzamide to the S-oxide plus a small
amount of benzamide (29). Under the same conditions, liver microsomes
convert the thiobenzamide S-oxide exclusively to benzamide.
This second reaction, which was proposed to involve conversion of the
thiobenzamide S-oxide to the unstable S,S-dioxide, appears to also be mediated by the
hepatic flavoprotein monooxygenase because it was inhibited by
inhibitors of that enzyme (29). Model studies show that thiobenzamide
is oxidized to the S-oxide and subsequently to the
S,S-dioxide by H2O2 (30)
or, much more rapidly, by a hydroperoxyflavin model of flavin
monooxygenases (31). The second oxidation, which was much slower with
H2O2 than the first S-oxidation,
produced the nitrile at pH >8, the benzamide at pH 4.0, and mixtures
of the two products at intermediate pH values (30). Thus, EtaA, like
the mammalian flavin monooxygenase, is capable of converting
thiobenzamide to the corresponding S-oxide and of further
oxidizing this intermediate to the amide.
In summary, Rv3854c codes for a flavoprotein containing a single FAD
group that catalyzes the NADPH- and
O2-dependent monooxygenation of ETA to the
corresponding S-oxide. This sulfoxidation is a required step
in the activation of ETA to the species directly responsible for the
antimycobacterial activity of the drug. EtaA is also capable of further
oxidizing ETA-SO to what is believed to be the final cytotoxic species.
This reaction does not occur until ETA-SO has appreciably accumulated
in the reaction mixture. We now plan to (a) explore the
further activation of ETA-SO to elucidate the nature of the putative
reactive metabolite of ETA and (b) express and characterize
the Rv3854c mutants known to convey resistance to ETA and to evaluate
their interaction with the drug. Elucidation of the role of the
mutations will make possible a better understanding of the mechanism of
action of flavoprotein encoded by Rv3854c.
We thank Clifton E. Barry III and Andrea E. DeBarber of the National Institutes of Health for the Rv3854c clone,
for information on its biological role ahead of publication (14), and
for initial reference samples of potential ETA metabolites. We thank
Eric B. Johansen of the B. Gibson Lab (National Institutes of Health Shared Instrumentation Program RR14601, University of California San
Francisco) for the mass spectral analyses.
*
This work was supported by National Institutes of Health
Grant GM56531.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: School of Pharmacy,
S-926, University of California, San Francisco, CA 94143-0446. Fax:
415-502-4728; E-mail: ortiz@cgl.ucsf.edu.
Published, JBC Papers in Press, January 31, 2002, DOI 10.1074/jbc.M110751200
The abbreviations used are:
INH, isoniazid;
ETA, ethionamide or 2-ethyl-4-thiocarbamoylpyridine;
ETA-SO, ethionamide
sulfoxide;
EtaA, ethionamide-activating enzyme;
HPLC, high-pressure
liquid chromatography;
ESI, electrospray ionization;
MS, mass
spectroscopy;
FPLC, fast protein liquid chromatography;
NTA, nitrilotriacetic acid.
The Antituberculosis Drug Ethionamide Is Activated by a
Flavoprotein Monooxygenase*
,, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The structures of ethionamide
(1), ethionamide S-oxide
(2), the postulated intermediate sulfinic acid species
(2a), 2-ethyl-4-cyanopyridine (3),
2-ethyl-4-amidopyridine (4), 2-ethyl-4-carboxypyridine
(5), and 2-ethyl-4-hydroxymethylpyridine
(6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C and was found to be stable for at
least 4 months at that temperature. HPLC columns were purchased from
Alltech Associates, Inc.
1 cm1. The
reaction medium contained 50 mM Tris buffer (pH = 7.5), 100 mM KCl, catalase (100 units/ml), superoxide
dismutase (100 units/ml), bovine serum albumin (0.1 mg/ml), and an
NADPH-regenerating system consisting of glucose-6-phosphate
dehydrogenase (2 units/ml), glucose 6-phosphate (250 mM),
and either NADP+ or NADPH. The final concentration of
enzyme was typically 500 nM in FAD. ETA stock solutions (25 mM) were freshly prepared in acetonitrile, and the final
added volume never exceeded 1% of the total reaction volume. All stock
solutions were kept on ice prior to mixing within the cuvette.
20 °C from hot chloroform layered with
diethyl ether yielded 172 mg (0.944 mmol) of a bright yellow
microcrystalline solid. This solid material is stored in the dark at
20 °C: 1H NMR
(d6-Me2SO, 400 MHz) [
(ppm)] 1.239 (3H, t, CH2CH3), 2.791 (2H, q, CH2CH3), 7.324 (1H, d,
5-H), 7.410 (1H, s, 3-H), 8.502 (1H, br s,
C(SO)NH2), 8.574 (1H, d, 6-H), 9.403 (1H, br s, C(SO)NH2); 13C NMR
(d6-Me2SO, 400 MHz) [ (ppm)]
13.481, 30.499, 116.119, 116.875, 136.476, 149.809, 163.967, 189.394;
UV-visible [
max (nm) (log(
) M1 cm1)] CH3CN,
365, and 50 mM Tris (pH = 7.5), 350 (3.78); ESI
IonTrap MS, observed m/z 183.13 (M + H)+, calculated for
C8H10N2SO 182.24.
1 (CN asymmetrical
stretch); 1H NMR
(d6-Me2SO, 400 MHz) [ (ppm)]
1.240 (3H, t, CH2CH3), 2.826 (2H, q,
CH2CH3), 7.667 (1H, d,
5-H), 7.767 (1H, s, 3-H), 8.736 (1H, d,
6-H); 13C NMR
(d6-Me2SO, 400 MHz) [ (ppm)]
13.266, 30.376, 116.989, 119.544, 122.738, 123.949, 150.147, 164.359 liters; UV-visible [max (nm)] 50 mM Tris
(pH = 7.5), 282; ESI IonTrap MS, observed
m/z 133.28 (M + H)+,
calculated for C8H8N2
132.18.
(ppm)]
1.251 (3H, t, CH2CH3), 2.808 (2H, q,
CH2CH3), 7.576 (1H, d,
5-H), 7.654 (2H, br s, 3-H and
NH2), 8.190 (1H, br s,
NH2), 8.596 (1H, d, 6-H);
13C NMR (d6-Me2SO, 400 MHz) [ (ppm)] 13.594, 30.617, 118.743, 119.636, 141.724, 149.450, 163.483, 166.541 liters; UV-visible [max (nm)] 50 mM Tris (pH = 7.5), 276; ESI IonTrap MS, observed
m/z 151.29 (M + H)+,
calculated for C8H10N2O
150.20.
(ppm)] 1.273 (3H, t,
CH2CH3), 3.024 (2H, q,
CH2CH3), 8.153 (1H, d,
5-H), 8.248 (1H, s, 3-H), 8.636 (1H, d,
6-H); UV-visible [max (nm)] 216; ESI
IonTrap MS, observed m/z 150.0 (M H), calculated for
C8H9NO2 151.18.
(ppm)] 7.8 (1H, m, para-H), 8.1 (2H, dd,
meta-H), 8.3 (1H, br s,
NH2), 8.4 (2H, dd,
ortho-H), 8.6 (1H, br s,
NH2); UV-visible [max (nm)] 50 mM Tris (pH = 7.4), 330 nm.
Escherichia coli
cells, and protein expression was induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside once the cells
grew to A600 = 0.9. Cells were grown for
24 h postinduction at 22 °C and were harvested by
centrifugation at 4,000 × g.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
[in a new window]
Fig. 2.
SDS-polyacrylamide gel of various stages of
purification of protein. Lane 1, crude lysate; lane
2, supernatant after lysis and sonication; lane 3, cell
pellet after lysis and sonication; lane 4, supernatant after
solubilization of membrane-bound proteins and centrifugation;
lane 5, pure protein after Ni-NTA affinity column.
Specific activities of the protein coded for by Rv3854c as a function
of purification stage
View larger version (18K):
[in a new window]
Fig. 3.
UV-visible spectra of extracted FAD
(solid line), authentic FAD (dashed
line), and purified EtaA (inset) in 50 mM Tris (pH = 7.4). 3X, 3-fold
y-axis amplification.
View larger version (25K):
[in a new window]
Fig. 4.
HPLC traces of 3-h incubation of EtaA with
200 µM ETA (top), 3-h incubation of EtaA with
200 µM ETA-SO (middle), and a mixture of 200 µM each of ETA, ETA-SO, 3, and 4 (bottom). Peak assignments are as follows
(tR, min): A, 4 (9.1);
B, ETA (9.7); C, ETA-SO (14.9); and
D, 3 (22.2). Chromatograms were recorded at 284 nm. mAU, milliarbitrary units.
1, and 7.73 (±5%) mol of ETA-SO min1
mol1, respectively (inset, Fig.
5). Spectroscopic analysis of the reaction of EtaA with ETA in the presence of an NADPH-regenerating system demonstrates a clean initial conversion of ETA to ETA-SO as
evidenced by a clear isosbestic point at 309 nm (Fig. 5).
View larger version (21K):
[in a new window]
Fig. 5.
UV-visible spectra of the formation of ETA-SO
from ETA during a 5-min incubation with EtaA. Inset,
double-reciprocal plot of initial rate measurements performed at
[ETA] = 50, 100, 200, and 400 µM. The initial reaction
system consisted of EtaA (500 nM), ETA (200 µM), and an NADPH-regenerating system comprised of NADPH
(200 µM), glucose-6-phosphate dehydrogenase (2 units/ml),
and glucose 6-phosphate (2.5 mM). The reaction was carried
out in 50 mM Tris (pH = 7.5) in the presence of
superoxide dismutase (100 units/ml), catalase (100 units/ml), bovine
serum albumin (0.1 mg/ml), and 100 mM KCl at
37 °C.
View larger version (10K):
[in a new window]
Fig. 6.
The structures of analogues of
ethionamide that are oxidized by the purified flavoprotein:
thiobenzamide (7) with its observed metabolite
thiobenzamide S-oxide (8),
isothionicotinamide (9), and thiacetazone
(10).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Both authors contributed equally to this work.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Farmer, P.,
Bayona, J.,
Becerra, M.,
Furin, J.,
Henry, C.,
Hiatt, H.,
Kim, J. Y.,
Mitnick, C.,
Nardell, E.,
and Shin, S.
(1998)
Int. J. Tuberc. Lung Dis.
2,
869-876[Medline]
[Order article via Infotrieve] 2.
Dye, C.,
Scheele, S.,
Dolin, P.,
Pathania, V.,
and Raviglione, M. C.
(1999)
J. Am. Med. Assoc.
282,
677-686 3.
Bass, J. B., Jr.,
Farer, L. S.,
Hopewell, P. C.,
O'Brien, R.,
Jacobs, R. F.,
Ruben, F.,
Snider, D. E., Jr.,
and Thornton, G.
(1994)
Am. J. Respir. Crit. Care Med.
149,
1359-1374[Abstract] 4.
Pablos-Mendez, A.,
Raviglione, M. C.,
Laszlo, A.,
Binkin, N.,
Rieder, H. L.,
Bustreo, F.,
Cohn, D. L.,
Lambregts-van Weezenbeek, C. S.,
Kim, S. J.,
Cahulet, P.,
and Nunn, P.
(1998)
N. Eng. J. Med.
338,
1641-1649 5.
Crofton, J.,
Chaulet, P.,
Maher, D.,
Grosset, J.,
Harris, W.,
Norman, H.,
Iseman, M,
and Watt, B.
(1997)
Guidelines for the Management of Multidrug-Resistant Tuberculosis
, World Health Organization, Geneva
6.
Banerjee, A.,
Dubnau, E.,
Quemard, A.,
Balasubramanian, V., Um, K. S.,
Wilson, T.,
Collins, D.,
de Lisle, G.,
and Jacobs, W. R.
(1994)
Science
263,
227-230 7.
Heym, B.,
Honore, N.,
Truffot-Pernot, C.,
Banerjee, A.,
Schurra, C.,
Jacobs, W. R., Jr.,
van Embden, J. D.,
Grosset, J. H.,
and Cole, S. T.
(1994)
Lancet
344,
293-298[CrossRef][Medline]
[Order article via Infotrieve] 8.
Wilson, M.,
DeRisi, J.,
Kristensen, H.-H.,
Imboden, P.,
Rane, S.,
Brown, P. O.,
and Schoolnik, G. K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
12833-12838 9.
Rist, N.,
Grumback, F.,
and Libermann, D.
(1959)
Am. Rev. Tuberc. Pulm. Dis.
79,
1-5
10.
Johnsson, K.,
and Schultz, P. G.
(1994)
J. Am. Chem. Soc.
116,
7425-7426[CrossRef] 11.
Johnsson, K.,
Froland, W. A.,
and Schultz, P. G.
(1997)
J. Biol. Chem.
272,
2834-2840 12.
Johnsson, K.,
King, D. S.,
and Schultz, P. G.
(1995)
J. Am. Chem. Soc.
117,
5009-5010[CrossRef] 13.
Rozwarski, D. A.,
Grant, G. A.,
Barton, D. H. R.,
Jacobs, W. R., Jr.,
and Sacchettini, J. C.
(1998)
Science
279,
98-102 14.
DeBarber, A. E.,
Mdluli, K.,
Bosman, M.,
Bekker, L.-G.,
and Barry, C. E., III
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9677-9682 15.
Baulard, A. R.,
Betts, J. C.,
Engohang-Ndong, J.,
Quan, S.,
McAdam, R. A.,
Brennan, P. J.,
Locht, C.,
and Besra, G. S.
(2000)
J. Biol. Chem.
275,
28326-28331 16.
Johnston, J. P.,
Kane, P. O.,
and Kibby, M. R.
(1967)
J. Pharm. Pharmacol.
19,
1-9[Medline]
[Order article via Infotrieve] 17.
Pütter, V. J.
(1972)
Arzneim.-Forsch.
22,
1027-1031 18.
Von, M.,
Grunert,
and Iwainsky, H.
(1967)
Arzneim.-Forsch.
17,
411-415 19.
Prema, K.,
and Gopinathan, K. P.
(1976)
J. Indian Inst. Sci.
58,
16-27
20.
Jenner, P. J.,
Ellard, G. A.,
Gruer, P. J. K.,
and Aber, V. R.
(1984)
J. Antimicrob. Chemother.
13,
267-277 21.
Bose, D. S.,
and Kumar, K. K.
(2000)
Synth. Commun.
30,
3047-3052
22.
Cacchi, S., Misiti, D., and La Torre, F. (1980)
Synthesis 243-244
23.
Van der Werf, M. J.
(2000)
Biochem. J.
347,
693-701 24.
Cashman, U. F.,
Parikh, K. K.,
Traiger, G. J.,
and Hanzlik, R. P.
(1983)
Chem.-Biol. Interact.
45,
341-347[CrossRef][Medline]
[Order article via Infotrieve] 25.
Cashman, J. R.,
and Hanzlik, R. P.
(1981)
Biochem. Biophys. Res. Commun.
98,
147-153[CrossRef][Medline]
[Order article via Infotrieve] 26.
Tynes, R. E.,
and Hodgson, E.
(1983)
Biochem. Pharmacol.
32,
3419-3428[CrossRef][Medline]
[Order article via Infotrieve] 27.
McManus, M. E.,
Stupans, I.,
Burgess, W.,
Koenig, J. A.,
Hall, P. M.,
and Birkett, D. J.
(1987)
Drug Metab. Dispos.
15,
256-261[Abstract] 28.
Hanzlik, R. P.,
Cashman, J. R.,
and Traiger, G. J.
(1980)
Toxicol. Appl. Pharmacol.
55,
260-272[CrossRef][Medline]
[Order article via Infotrieve] 29.
Hanzlik, R. P.,
and Cashman, J. R.
(1983)
Drug Metab. Dispos.
11,
201-205[Abstract] 30.
Cashman, J. R.,
and Hanzlik, R. P.
(1982)
J. Org. Chem.
47,
4645-4650[CrossRef] 31.
Doerge, D. R.,
and Corbett, M. D.
(1984)
Mol. Pharmacol.
26,
348-352[Abstract]
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