(Received for publication, July 13, 1995)
From the
The unique antitubercular activity of isoniazid requires that
the drug be oxidized by the katG-encoded mycobacterial
catalase-peroxidase to an activated drug form. In order to
quantitatively assess the catalytic capabilities of the enzyme, the
native catalase-peroxidase from Mycobacterium smegmatis was
purified over 200-fold to homogeneity. The enzyme was shown to exhibit
both catalase and peroxidase activities, and in the presence of either
hydrogen peroxide or t-butyl peroxide, was found to catalyze
the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as
well as artificial peroxidase substrates, at rates between 2.7 and 20
s. The homogeneous enzyme exhibited a visible
absorbance spectrum typical of ferric heme-containing
catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature
(10 K) electron paramagnetic resonance spectra in the presence of
ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t =
20 min) reduced by dithionite, and the reduced form
showed typical spectral changes when either KCN or CO were subsequently
added. The M. smegmatis catalase-peroxidase was found to
contain 2 heme molecules per tetramer, which were identified as iron
protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic
activity was inhibited by KCN, NaN
, isoniazid (isonicotinic
acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by
3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases
in the oxidative activation of the antitubercular prodrug isoniazid is
discussed.
Metabolism in an oxygen-containing environment results in the generation of reactive oxygen species such as superoxide, hydroxyl radical, and hydrogen peroxide. Organisms have evolved sophisticated and efficient enzyme systems to neutralize these potentially injurious reactive oxygen species, including catalases, superoxide dismutases, and peroxidases. Enzymes that exhibit either catalatic or peroxidatic activity are common among animals, plants, and microorganisms, while bacteria and some fungi have been found to contain hydroperoxidases that exhibit both catalase and peroxidase activities. These catalase-peroxidases have been purified from Escherichia coli B(1) , Klebsiella pneumoniae(2) , Halobacterium halobium(3) , Septoria tritici(4) , and a Bacillus strain YN-2000(5) , and exhibit visible absorbance spectra typical of a ferric high spin hemoprotein. The pyridine ferrohemochromes of these enzymes show an absorption spectrum typical of ferric protoheme IX(1, 2, 3, 4, 5) . A characteristic feature of the bacterial catalase-peroxidases is their low heme content, with only 2 molecules of heme per enzyme tetramer. The activity of bacterial catalase-peroxidases is inhibited by azide and cyanide, but not by the catalase-specific inhibitor, 3-amino-1,2,4-triazole.
The enzymological characterization of
mycobacterial catalase-peroxidases is relevant for a number of reasons.
Human mycobacterial pathogens, such as Mycobacterium
tuberculosis, Mycobacterium avium, and Mycobacterium
leprae are phagocitized by, and can proliferate inside,
macrophages, and catalase-peroxidases help to protect the bacilli from
the deleterious effects of macrophage-generated hydrogen
peroxide(6) . kat gene-encoded proteins have been
shown to be related to the survival of different bacteria under
oxidative stress conditions. In E. coli, both a constitutively
expressed katE-encoded catalase and a hydrogen
peroxide-inducible katG-encoded catalase-peroxidase are
present(7) , while in M. tuberculosis the katG-encoded catalase-peroxidase is constitutively expressed
and no katE-type catalase has been identified(8) . In
the nonpathogenic M. smegmatis, there appear to be both katE- and katG-type catalases that are expressed
under a variety of conditions(9) , although our current
interest concerns only the properties of the latter. The M.
tuberculosis catalase-peroxidase (10) has been implicated
in the susceptibility of the organism to isoniazid, and in the
mechanism of isoniazid (INH) ()action, since isoniazid drug
resistance is correlated with the loss of catalase-peroxidase
activity(11) . More recently, it has been demonstrated that the
deletion of, or point mutations in, the M. tuberculosis
katG-encoded catalase-peroxidase results in high level INH
resistance, and that transformation of INH-resistant strains with a
functional M. tuberculosis katG gene restores sensitivity to
the drug(12, 13) . Moreover, the virulence of Mycobacterium bovis in guinea pigs has been shown to be
dependent on katG. INH-resistant strains, lacking katG, were significantly less virulent than the parent
INH-sensitive strains, and integration of a functional katG
gene into the most resistant strain restored full
virulence(14) .
It has been demonstrated that the M. tuberculosis catalase-peroxidase catalyzes the oxidation of isoniazid, resulting in the formation of reactive intermediates that react with added nucleophiles(15) . Furthermore, the catalase-peroxidase-activated isoniazid binds to and inhibits the activity of the mycobacterial fatty acyl enoyl-ACP reductase encoded by the inhA gene(16) , the enzyme considered to be the target of isoniazid action(17) . Although many mycobacterial katG genes have been cloned and sequenced, few have been characterized, and little information about the catalytic properties of the enzyme has appeared, even in light of the central role of the enzyme in isoniazid activation.
In this report, we describe the purification of a constitutively expressed catalase-peroxidase from M. smegmatis and characterize both the spectroscopic properties of the enzyme and the catalytic reactions.
M. smegmatis mc155 cells (60-80 g
wet weight) were suspended in 50 mM TEA buffer, pH 7.8 (50%
w/v), containing protease inhibitors (typically, per liter, 2.3 mg of
leupeptin, 52 mg of 1-chloro-3-tosylamido-7-amino-2-heptadone, 20 mg of
soybean trypsin inhibitor, 1.6 mg of aprotinin, 1.1 mg of pepstatin,
and 36.2 mg of phenylmethylsulfonyl fluoride). Cells were broken by
sonication, and cell debris was removed by centrifugation for 45 min at
12,000 rpm. Nucleic acids were precipitated by the addition of
streptomycin sulfate (1% w/v, final concentration) to the supernatant,
and the solution was centrifuged for 45 min at 12,000 rpm to pellet the
nucleic acids. The supernatant was dialyzed against 20 mM TEA
buffer, pH 7.8, for 3 h, during which time a precipitate forms which
was removed by centrifugation at 45,000 rpm for 45 min. The clear
supernatant was applied to a 400-ml Fast-Flow Q-Sepharose (Pharmacia
Biotech Inc.) anion-exchange column which had been equilibrated with 20
mM TEA, pH 7.8. The adsorbed protein was eluted using a
2-liter nonlinear 0-1 M NaCl gradient. Fractions
containing peroxidatic activity eluting between 0.3 and 0.4 M NaCl were pooled, concentrated (PM 10, Amicon) to 5 ml, and
applied to a 1.6
60-cm Superdex 200 (Pharmacia) gel filtration
column equilibrated with 20 mM TEA, pH 7.8, containing 50
mM NaCl. The active fractions were pooled, dialyzed, and
applied to a 1
10-cm Mono Q (Pharmacia) high performance
anion-exchange column. The enzyme was eluted using a 200-ml nonlinear
0-0.6 M NaCl gradient. The active fractions, eluting
between 0.2 and 0.3 M NaCl, were pooled, made 1 M in
ammonium sulfate by addition of solid ammonium sulfate, and applied to
a phenyl-Superose (Pharmacia) column. The protein was eluted with a
200-ml reverse gradient of ammonium sulfate (1-0 M) in
20 mM TEA, pH. 7.8, and the active fractions were pooled. The
enzyme solution was brownish, and displayed a single band of
80,000 daltons on SDS-PAGE with Coomassie Blue staining (data not
shown).
The concentration of enzyme active sites was calculated
based on the pyridine hemochromogen assay, using = 191.5 mM
cm
(18) . The purified protein (200
pmol) was analyzed by electrospray ionization/mass spectrometry in an
API III triple-quadrupole mass spectrometer (PE SCIEX). The data were
deconvoluted by computer to determine the subunit molecular weight.
Automated amino-terminal sequencing was performed using an Applied
Biosystem sequencer using standard Edman chemistry.
The peroxidase
activity of the enzyme was determined spectrophotometrically by
measuring the rate of oxidation of 0.1 mMo-dianisidine at 460 nm ( =
11.3 mM
cm
) in the
presence of 23 mMt-butyl hydroperoxide. Catalase
activity was determined polarographically using a Clark electrode
(Orbisphere Laboratories, Geneva) by measuring the formation of
molecular oxygen from H
O
. The oxygen electrode
was calibrated in air saturated water at 25 °C, assuming the
concentration of dissolved oxygen under these conditions is 256
µM.
Low temperature EPR spectroscopy was performed using a Varian E-112 X-band spectrometer equipped with a Varian NMR gaussmeter and a Systron-Donner frequency counter. Samples were maintained at approximately 10 K using a Heli-Tran liquid helium transfer system (Air Products). Spectral data were collected using software provided by the ESR Center of the University of Illinois(19) . Experimental parameters are given in the figure legends.
Kinetic parameters were determined in 50 mM
phosphate buffer, pH 7.0, at 25 °C by following NAD(P)H oxidation
spectrophotometrically at 340 nm ( = 6.2
mM
cm
),
2,2`-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation at 414
nm (
= 36 mM
cm
), pyrogallol oxidation at 430 nm
(
= 2.47 mM
cm
), and 3,3`-diaminobenzidine oxidation at
416-nm (
= 5.68 mM
cm
) using a thermostated Uvicon 9310
spectrophotometer. The concentrations of the substrates were calibrated
enzymatically using excess H
O
and horseradish
peroxidase. The concentration of peroxides was determined
iodometrically, using iodide in the presence of horseradish peroxidase
and limiting amounts of peroxide in acetate buffer, pH 3.8. The
absorbance changes were monitored spectrophotometrically at 353 nm,
using
= 25.5 mM
cm
(20) for
I
.
Initial velocity studies were performed by varying the reducing substrate concentration at several fixed concentrations of alkyl or hydrogen peroxide. Data were plotted in reciprocal form, and fitted to , using the Fortran programs of Cleland(21) :
Inhibition studies were performed by varying the concentration of inhibitor versus one of the substrates at a fixed, saturating concentration of the other substrate. Data were plotted in reciprocal form, and fitted to , which describes linear, competitive inhibition:
Figure 1: Absorption spectrum of purified catalase-peroxidase from M. smegmatis.
Figure 2: Absorption spectrum of the CO complex formed with the reduced catalase-peroxidase in 50 mM phosphate buffer, pH 7.0, 25 °C. Time intervals between the scans were 3 min after addition of dithionite in the presence of CO. (Dotted line, oxidized enzyme.)
The pyridine ferrohemochrome
prepared from the enzyme showed a visible absorption spectrum typical
for protoheme IX, with peaks at 418, 523, and 555 nm(18) . The
protoheme content in the enzyme was estimated to be 2.0 ± 0.2
molecules per 330,000-dalton tetramer, based on the pyridine
ferrohemochrome absorbance at 418 nm. This low heme content is
consistent with the low A/A
ratio of 0.56 for the purified protein (Fig. 1). These
properties are similar to those previously reported for other bacterial
catalase-peroxidases (1, 2, 3, 4, 5) .
Figure 3: EPR spectra of M. smegmatis catalase-peroxidase. Inset at top shows the x-band EPR spectrum of the resting enzyme recorded at 10 K. A, low field region of spectrum in inset; B, low field region after addition of excess of NaF to sample in A. Experimental conditions: microwave power, 10 mW; modulation amplitude, 10 G; microwave frequency, 9.308 GHz. A baseline correction was performed digitally for the data in B.
An EPR
signal could also be elicited by the addition of sodium fluoride to the
enzyme sample in TEA buffer, without the addition of ethylene glycol (Fig. 3B). This signal, which was the same in the
presence of ethylene glycol (not shown), also had a very small rhombic
splitting though the g values (g = 6.1, 5.8) were different from
those observed in the presence of ethylene glycol alone. No
identification of features at g = 2.0 could be made (nor could a
splitting due to F hyperfine interaction be resolved)
although fluoride binding is evident from the observed change in g
values.
In addition to the
peroxidatic activity, the enzyme exhibited catalatic activity, which
was comparable to analogous enzymes activities. The catalatic activity
of the mycobacterial enzyme is significantly higher than that exhibited
by the E. coli catalase-peroxidase (2380 s compared to 563 s
(1) , respectively,
at pH 7.5), although the K
values for
H
O
are comparable for these two enzymes (1.43
and 3.9 mM, respectively). The V/K
value of the catalatic reaction catalyzed by the mycobacterial
catalase-peroxidase (1.7
10
M
s
) is an order of magnitude lower than that
reported for bovine liver catalase (2.1
10
M
s
)(24) .
Figure 4:
Isoniazid inhibition versus
o-dianisidine. 50 mM phosphate buffer, pH 7.0, 25 °C.
The fit of the data yield a calculated K = 4.3 ± 0.7
µM.
A wide variety of bacteria have been shown to express heme-containing hydroperoxidases capable of performing both catalatic and peroxidatic chemistries(1, 2, 3, 4, 5) . The katG-encoded catalase-peroxidases from several mycobacterial species have been cloned and sequenced, and the M. intracellulare katG gene has been expressed in E. coli and shown to exhibit both catalase and peroxidase activities(22) . However, no detailed substrate specificity studies, nor spectroscopic characterization, has been performed on any mycobacterial catalase-peroxidase. Our interest in these enzymes stems from the unique role of the enzyme in oxidative stress management, virulence and the unique susceptibility of mycobacteria, especially M. tuberculosis, to isoniazid.
Pathogenic mycobacteria,
including M. tuberculosis, M. leprae, and the M.
avium-M.intracellulare complex, are phagocytized by
macrophages and can survive and proliferate inside macrophage
phagosomes. One of the reasons mycobacteria can survive the high levels
of reactive oxygen species, particularly HO
,
generated by the macrophage, is that they can efficiently remove this
injurious oxidant by the action of intracellular catalases and
peroxidases. M. tuberculosis contains only a constitutively
expressed katG-encoded catalase-peroxidase(8) , while
other mycobacteria may contain an additional katE-type
catalase (9) , as does E. coli(7) .
Mycobacterial catalase-peroxidases (katG-encoded proteins) are
highly homologous, and are additionally homologous to other bacterial
catalase-peroxidases. Though a certain amount of structural information
is appearing for these enzymes, little is known about their catalytic
properties. The paucity of mechanistic information is surprising
considering the critical role of the mycobacterial catalase-peroxidase
in the action of the potent antitubercular drug, isoniazid (see below).
This encouraged us to undertake the purification and characterization
of the constitutively expressed catalase-peroxidase from M.
smegmatis.
The enzyme purified from M. smegmatis was
found to share a number of structural and spectroscopic properties with
other bacterial catalase-peroxidases. It is a tetramer of 81,889-dalton
subunits, containing two ferric protoporphyrin IX prosthetic groups,
and exhibits a high spin ferric heme optical spectrum. The iron could
be slowly reduced by dithionite and converted to a low spin ferrous
form in the presence of either CN or CO. Dithionite
reduction distinguishes the catalase-peroxidases from the typical
catalases, which are not reduced by dithionite, and typical
peroxidases, which are rapidly reduced by dithionite. The rate of
dithionite reduction is atypical for both catalases and peroxidases.
The EPR results indicate that ethylene glycol stabilizes high spin ferric heme iron in the catalase-peroxidase enzyme. The absence of EPR signals in frozen solutions without added ethylene glycol or fluoride ion may arise from a freezing artifact similar to that previously described for two other heme peroxidases, cytochrome c peroxidase (25) and lactoperoxidase(26) . In these cases, freezing induces the formation of low spin species, which for cytochrome c peroxidase has been reported to involve coordination of an endogenous strong field ligand(25) . The formation of a mixture of species upon freezing dilute samples of catalase-peroxidase in buffer alone could similarly be responsible for the loss of resolvable intensity of either high or low spin forms.
The EPR signal observed in ethylene glycol/buffer mixtures are
atypical for classical catalases, such as bovine liver catalase (27) and plant or bacterial peroxidases such as horseradish
peroxidase (28) and cytochrome c peroxidase(29) , all of which exhibit larger rhombic
splittings, with the lowest field g value ranging from 6.6 to 6.3. In
contrast to these, plant lignin peroxidase (30) and high spin
globins (27) exhibit no rhombic splitting and a g value near
5.8. The EPR spectrum of catalase-peroxidase from Streptomyces has recently been reported, although the multiple g features at g
5-7 make comparison to our results
difficult(31) . The small rhombicity found for the M.
smegmatis catalase-peroxidase, usually encountered for high spin
globins(27) , is considered to be an indicator of the presence
of six-coordinated ferric heme containing a proximal imidazole as the
fifth ligand to iron and water as sixth ligand. This idea is supported
by alignment of the amino acid sequences of cytochrome c peroxidase (for which a high resolution crystal structure is
available), bacterial catalase-peroxidases (32) and
mycobacterial catalase-peroxidases(22, 33) . There is
a highly conserved region in the NH
-terminal domain
(RMGMNDEETVALIAGGHTLGKTH), and the underlined residue His-175 of
cytochrome c peroxidase is a proximal ligand of the heme iron
and is conserved in other catalase-peroxidases (His-267 in E.
coli, His-269 in M. tuberculosis, and His-277 in M.
intracellulare). This homology is not apparent in the
amino-terminal region where, with the exeption of the M.
intracellulare (Table 2), divergent sequences are observed
between bacterial catalase-peroxidases and especially between
catalase-peroxidases and cytochrome c peroxidase, although
these differences were not considered catalytically significant, as
judged by activity staining(34) .
Additional characteristics
shared with other bacterial catalase-peroxidases include the inhibition
by CN and N
and the
lack of inhibition by the catalase inhibitor,
3-amino-1,2,4-triazole(35) . The linear, competitive nature of
the inhibition by CN
versus peroxide
suggests that the heme iron atom of the catalase-peroxidase is the site
of both peroxide and cyanide binding, as is the case for other
peroxidases(36) .
In contrast to cyanide inhibition, azide was found to be a linear, competitive inhibitor versus o-dianisidine, suggesting that it binds at the reducing substrate binding site of catalase-peroxidase. It has been proposed that electron donors bind to the enzyme at a hydrophobic site 7-8.5 Å from the heme iron(37) . In horseradish peroxidase, azide inhibits the enzymatic activity yet does not bind at neutral pH to the prosthetic heme iron atom(38) . The one-electron oxidation of azide is catalyzed by chloroperoxidase, myeloperoxidase, and lactoperoxidase(39) , and the inhibition of these enzymes, as well as the mycobacterial catalase-peroxidase, may be due to heme modification by the product of the one-electron oxidation of azide.
Like other peroxidases and catalase-peroxidases, the mycobacterial enzyme oxidized artificial electron donors. However, its ability to use both NADH and NADPH as reductants is a unique property of bacterial catalase-peroxidases (2, 4) and distinguishes this class of hydroperoxidases from classical peroxidases and catalases. It is likely that reduced pyridine nucleotides are the in vivo reductants for the mycobacterial catalase-peroxidases, and the enzyme may thus serve physiologically to oxidize the reduced coenzymes, influencing the rates of metabolic pathways which are sensitive to this ratio. In contrast, mammalian catalases have been shown to contain four tightly bound molecules of NADPH(40) , although the function of the bound nucleotide is not clear. It has been proposed that the bound nucleotide protects the enzyme from inactivation, both by preventing, and reversing, the formation of compound II, an inactive form of catalase(40, 41) .
Perhaps the most intriguing feature of the mycobacterial catalase-peroxidases is their role in the activation of the antitubercular drug, isoniazid. There is compelling evidence that the antimycobacterial activity of isoniazid is dependent on the activity of the catalase-peroxidase(42, 43) . Isoniazid resistance in mycobacteria is correlated with lowered or absent enzymatic activity(12, 13) , and missense mutations in the amino-terminal peroxidase domain of the protein result in reduced enzyme activity and increased isoniazid resistance for strains expressing these mutated enzymes(44, 45) .
Our inhibition results suggest that isoniazid, in addition to being
an oxidizable substrate for the M. tuberculosis(15) and M. smegmatis catalase-peroxidase, ()is also a potent, competitive inhibitor of the peroxidatic
reaction, competing for the substrate binding site. The lack of
inhibition using other aromatic ring containing inhibitors (Table 4) suggests that the hydrazide group is critical for the
activity of the compound. Isoniazid will thus inhibit the normal,
protective functions of the enzyme in peroxide reduction, and will
enhance the bactericidal activity of macrophage-generated peroxide.
Equally importantly, the product of the catalase-peroxidase catalyzed
oxidation of isoniazid (15) both binds to, and inhibits the
activity of, the mycobacterial fatty enoyl-ACP reductase(16) .
This enzyme, encoded by the inhA gene(46) , has been
shown to be involved in the biosynthesis of long chain mycolic acids
found in mycobacterial cell walls(17) . It is interesting to
note that nicotinic acid hydrazide is oxidized by the M.
tuberculosis katG-encoded catalase-peroxidase at rates comparable
to those observed for isoniazid(15) , although the former
compound exhibits no antitubercular activity(42) . These
results are in agreement with our finding that both isoniazid and
nicotinic acid hydrazide bind to, and inhibit the peroxidatic reaction
of both the M. smegmatis and M. tuberculosis catalase-peroxidase to the same extent (Table 4).
The 10-fold lower peroxidatic activity of the M. tuberculosis catalase-peroxidase compared to that of M. smegmatis is unexpected, since if sensitivity to isoniazid only involves rates of conversion of the prodrug, isoniazid, into an activated form, then one would expect that M. smegmatis would be more sensitive to isoniazid than M. tuberculosis. This is clearly not the case, since reported values for the minimal inhibitory concentration of isoniazid are 0.02 and 8 µg/ml for M. tuberculosis H37Rv and M. smegmatis, respectively(47, 48) . Possible reasons for the greater sensitivity of M. tuberculosis to isoniazid may be related to prodrug uptake, target sensitivity to inhibition, or other interspecies differences. These possibilities are under investigation.