Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles

Article abstract of DOI:10.1111/j.1742-4658.2011.08404.x

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F₄₂₀) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F ₄₂₀. The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.

Full text of DOI:10.1111/j.1742-4658.2011.08404.x

Substrate specificity of the deazaflavin-dependent
nitroreductase from Mycobacterium tuberculosis
responsible for the bioreductive activation of bicyclic
nitroimidazoles
1
2
2,
2,
Meera Gurumurthy , Tathagata Mukherjee , Cynthia S. Dowd *, Ramandeep Singh ꢀ, Pornwaratt
1
1,
2
3
1,
Niyomrattanakit , Jo Ann Tay ꢁ, Amit Nayyar , Yong Sok Lee , Joseph Cherian §,
Helena I. Boshoff , Thomas Dick –, Clifton E. Barry III and Ujjini H. Manjunatha
2
1,
2
1
1
2
3
Novartis Institute for Tropical Diseases, Singapore, Singapore
Tuberculosis Research Section, NIAID, NIH, Bethesda, MD, USA
Center for Molecular Modeling, Center for Information Technology, NIH, Bethesda, MD, USA
Keywords
The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promis-
ing novel class of therapeutics for tuberculosis and are currently in phase
II clinical development. Both compounds are pro-drugs that are reductively
activated by a deazaflavin (F420) dependent nitroreductase (Ddn). Herein
we describe the biochemical properties of Ddn including the optimal enzy-
matic turnover conditions and substrate specificity. The preference of the
enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the
presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only
short alkyl substituents at the C-7 position of the oxazole were reduced by
Ddn without any stereochemical preference. However, with bulkier substi-
tutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn
mediated metabolism of PA-824 results in the release of reactive nitrogen
species. We have employed a direct chemiluminescence based nitric oxide
(NO) detection assay to measure the kinetics of NO production by Ddn.
Binding affinity of PA-824 to Ddn was monitored through intrinsic fluores-
cence quenching of the protein facilitating a turnover-independent assess-
ment of affinity. Our results indicate that (R)-PA-824, despite not being
turned over by Ddn, binds to the enzyme with the same affinity as the
active (S) isomer. This result, in combination with docking studies in the
active site, suggests that the (R) isomer probably has a different binding
mode than the (S) with the C-3 of the imidazole ring orienting in a non-
F420; mycobacterium; nitroimidazole;
PA-824; tuberculosis
Correspondence
U. H. Manjunatha, Novartis Institute for
Tropical Diseases, 10 Biopolis Road,
Singapore 138670
Fax: +65 6722 2917
Tel: +65 6722 2976
E-mail: manjunatha.ujjini@novartis.com
C. E. Barry III, Tuberculosis Research
Section, NIAID, NIH, Bethesda, MD 20892,
USA
Fax: +1 301 480 5705
Tel: +1 301 435 7509
E-mail: cbarry@niaid.nih.gov
Present addresses
*
George Washington University,
Washington, DC, USA
ꢀ
ꢁ
THSTI, Gurgaon, India
SIgN, Singapore
§ETC, Singapore
–
National University of Singapore, Singapore productive position with respect to the incoming hydride from F420. The
results presented provide insight into the biochemical mechanism of reduc-
tion and elucidate structural features important for understanding substrate
binding.
M. G. and T. M. contributed equally to this
work
(Received 7 September 2011; revised 13
October 2011; accepted 13 October 2011)
doi:10.1111/j.1742-4658.2011.08404.x
Abbreviations
Ddn, deazaflavin-dependent nitroreductase; FGD1, F420-dependent glucose-6-phosphate dehydrogenase; MIC, minimal inhibitory
concentration; Mtb, Mycobacterium tuberculosis; Mtz, metronidazole; PMT, photomultiplier tube; TB, tuberculosis; WT, wild-type.
FEBS Journal 279 (2012) Journal compilation 113–125 ª 2011 FEBS. No claim to original US government works
113

Biochemical characterization of Mtb Ddn
M. Gurumurthy et al.
Introduction
One-third of the human population is infected with
tuberculosis (TB) and nearly 2 million die every year
from active disease [1]. Mycobacterium tuberculosis
identify the cellular machinery involved in its activa-
tion. The original observation that the F420-dependent
glucose-6-phosphate dehydrogenase (FGD1) was
required for sensitivity [14] was followed by a series of
papers describing resistance in isolates that had lost
the ability to biosynthesize the deazaflavin cofactor
F420 [17,18] and culminated in identification of a con-
served hypothetical protein encoded by Rv3547 that
was essential for susceptibility to the compound [19].
FGD1 catalyzes the oxidation of glucose-6-phosphate
to phosphogluconolactone and in turn reduces F420 to
(Mtb), the causative agent of TB, invades human alve-
olar macrophages where it has evolved to evade or sur-
vive the hostile conditions within these professional
phagocytes [2]. Partial containment of infection is
accomplished by formation of a highly organized, mul-
ticellular granuloma within which nutrient deprivation
and hypoxia contain, but do not kill, the invading
microbe [3]. Perhaps because of the complexity of the
intracellular environment and the variable replication
state of the organism, current treatment for TB
requires a combination of isoniazid, rifampicin, pyrazin-
amide and ethambutol for 2 months followed by isoni-
azid and rifampicin for 4 months [4]. Inadequate
treatment and failure to comply with drug regimens
have resulted in the emergence of multi-drug resistant
and extensively drug resistant Mtb strains [5,6]. New
agents with the potential to shorten treatment, for
example by killing anaerobically non-replicating Mtb,
are urgently needed to reverse current trends.
F420H . F420, an obligate two electron transfer agent,
2
is a low redox potential, soluble 7,8-didemethyl-8-
hydroxy-5-deazariboflavin with a ribosyl-phospholactyl
moiety and polyglutamate chain (Fig. 1) [20]. F420H is
2
the active form of the cofactor that is utilized by a
protein encoded by Rv3547, an enzyme henceforth
referred to as a deazaflavin-dependent nitroreductase
(Ddn). The physiological role of Ddn is unknown. The
F420H -dependent reduction of PA-824 by Ddn pro-
2
duces three stable metabolites resulting from reduction
of the imidazole ring at C-3 [21]. One of the major
products formed is the des-nitro metabolite with subse-
quent release of nitrous acid which degrades to NO.
We have evaluated several PA-824 analogs and
observed that the amount of des-nitro metabolite
formed (and the corresponding amount of NO gener-
ated) correlates well with their anaerobicidal activity
[21]. However, the aerobicidal and anaerobicidal activi-
ties of PA-824 correlate poorly [22,23] and our mecha-
nistic understanding of the reaction course leading to
aerobic or anaerobic activity remains incomplete.
Nitroimidazoles are commonly used antibiotics for
anaerobic microbial infections in humans. They show
better activity against obligate anaerobes than aerobic
organisms because their bactericidal activity requires an
oxygen-sensitive bioreduction of the aromatic nitro
group [7]. Metronidazole (Mtz) is a widely used 5-nitro-
imidazole for the treatment of infections caused by
anaerobic bacteria [8,9]. Mtb cultures become sensitive to
Mtz under low oxygen conditions. However, the com-
pound has no activity against aerobically growing
bacteria [10]. Aerobic antitubercular activity of bicyclic
nitroimidazoles was first reported from a series of 4- and
In order to further enhance our understanding of
the mechanism of action of PA-824, we have studied
the F420H -dependent nitroreductase activity of Ddn
5-nitroimidazole[2,1-b]oxazoles [11]. A highly active lead
2
compound, CGI-17341, showed activity in a murine
infection model but was subsequently dropped [12,13]
because of in vitro mutagenic results in the Ames test.
Subsequent elaboration of the bicyclic nitroimidazole ser-
ies resulted in the identification of Ames negative analogs
PA-824 [14] and OPC-67683 [15], two compounds that
are currently in phase II clinical development (Fig. 1).
Both compounds are pro-drugs that require bioreductive
activation and show no cross-resistance to other front-
line TB drugs. Activated PA-824 inhibits lipid biosynthe-
sis in a dose-dependent manner [14]. Transcriptional pro-
filing analyses of PA-824 treated cells suggest that
inhibition of both cell wall biosynthesis and respiration
contribute to the cidal activity of this compound [16].
Several studies have characterized spontaneously
generated PA-824 resistant mutants as a means to
using PA-824 and a selected collection of chemically
distinct nitroimidazole analogs by investigating reoxi-
dation of F420H , production of NO and by determin-
2
ing binding constants of the analogs to the protein.
These results suggest that the tail portion of the nitro-
imidazole determines the binding orientation of the
head group, conferring stereospecificity in orientation
of the molecule towards reduction.
Results
Optimization of Ddn catalyzed PA-824 reduction
Recombinant Ddn was expressed and purified as an
MBP-His6 tagged protein in Escherichia coli (see
Experimental procedures). F420 was isolated from
1
14
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M. Gurumurthy et al.
Biochemical characterization of Mtb Ddn
Fig. 1. Chemical structures of F420 and nitroimidazole substrates used in this study. The C-5 carbon of F420 carrying hydride and the pro-
posed site of initial hydride addition on C-3 of PA-824 are each shown by an asterisk.
Mycobacterium smegmatis and reduced to F420H2 as
described in Experimental procedures. Enzymatic
activity of Ddn was determined spectrophotometrically
by monitoring F420H2 oxidation at A400 nm. The
enzyme activity of Ddn was determined between pH
presence of various other redox active cofactors. The
enzyme was not able to utilize NADH and NADPH in
the reduction of PA-824 and was highly specific for
F420H (data not shown). F is present within cells as
2
420
a series of polyglutamylated forms containing two to six
glutamates in the tail in a species-specific pattern. Both
M. smegmatis and Mtb produce predominantly F420
species with five to six glutamate residues (Fig. 1) [24].
The ability of Ddn to utilize the F420 isolated from
Methanobacterium thermoautotrophicum, which produ-
ces F420 with two glutamate residues in its side chain
(F420-2) [25], was therefore evaluated. Ddn enzyme was
able to utilize reduced F420-2 (Km,app 26 lm) as effi-
ciently as F420-5 from M. smegmatis (Km,app 29 lm).
6.0 and pH 8.0; the optimum pH for Ddn mediated
F420H oxidation was 7.0–8.0 (Fig. S1D). At pH 8.0,
the enzyme was active at ambient temperature whereas
2
its activity was compromised at 48 ꢀC (Fig. S1E). The
inclusion of various monovalent (Na , K ) and diva-
+
+
2
+
2+
lent ions (Mg , Mn ) had no effect on enzyme
activity (data not shown). However, the presence of a
detergent (0.01% Triton X-100) improved the enzyme
activity significantly; thus the final buffer contained
2
00 mm Tris ⁄ HCl (pH 8.0) with 0.01% Triton X-100
Fig. S1F). Addition of 10% dimethylsulfoxide
(
(
The lipophilic tail of nitroimidazole substrates
determines reduction selectivity and efficiency
DMSO), 2 mm dithiothreitol (DTT) and 20 mm
EDTA to the Ddn buffer had no effect on enzyme
activity (Fig. S1G).
To understand the effect of substrate structure on the
enzyme kinetic parameters, we first determined the
apparent steady state kinetic constants Km,app and
The F420H2 specificity of Ddn was investigated by
monitoring Ddn catalyzed PA-824 reduction in the
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115

Biochemical characterization of Mtb Ddn
M. Gurumurthy et al.
kcat ⁄ Km,app for the Ddn reaction with PA-824 in the
and (S) stereoisomers of a simple phenyloxazole series
(Fig. 1). Ddn was not able to oxidize F420H2 when
using des-nitro PA-824 or (R)-PA-824 as substrates
(Fig. 2B), supporting the importance of the nitro
group and the previously noted enantiomeric specificity
for reduction of these nitroimidazo-oxazines [21].
Ddn showed no biochemical activity using Mtz, a
monocyclic 5-nitroimidazole, as a substrate (Fig. 2B)
presence of 100 lm F420H and 1 lm Ddn (Fig. 2A,B).
2
The K
0
for PA-824 was 29 lm and kcat ⁄ Km,app was
)1
m,app
)1
.16 lm Æmin (Fig. 2B, Table 1). To explore reduc-
tion selectivity, we determined the kinetic parameters
of Ddn with a limited set of structurally diverse PA-
824 analogs including (R)-PA-824, des-nitro PA-824,
Mtz, (R) and (S) stereoisomers of CGI-17341 and (R)
Fig. 2. Ddn kinetics monitored via F420
absorbance at 400 nm. (B) Michaelis–Menten plots for the Ddn catalyzed reduction of PA-824, (R)-PA-824, des-nitro PA-824, metronidazole,
Aza- PA-824, (R)- and (S)-CGI-17341 and (R)- and (S)-phenyloxazole derivatives at 100 lM F420 . Each value represents the mean ± standard
deviation of the initial velocity of formation of oxidized F420 from a linear regression analysis. (C), (D) Two-substrate profile analysis for Ddn.
Michaelis–Menten plot of (C) initial velocity versus [PA-824] and (D) initial velocity versus [F420 ] in the presence of 1 lM Ddn and varying
2 2
H oxidation. (A) Oxidation of F420H at varying PA-824 concentrations monitored by the increase in
H
2
H
2
concentration of the other substrate. Each value represents the mean ± standard deviation of the initial velocity of formation of oxidized F420
from a linear regression analysis. (E) Schematic representation of Ddn reaction mechanism: progression of reaction via a ternary complex
2
Ddn : F420H : PA-824 to form F420 and PA-824 metabolite products.
Table 1. Characterization of various PA-824 analogs. MIC, minimal inhibitory concentration; ns, not a substrate.
F
420
H
2
oxidation assay
MIC (lM)
Mtb WT
)1
cat ⁄ Km,app (lM)1Æmin)1
a
)
b
Mtb : Ddn
)
K
m,app (lM)
k
cat,app (min
)
k
)
Mtb : F420
PA-824
28.6 ± 3.6
ns
4.7 ± 0.3
ns
0.160 ± 0.020
ns
0.8
> 100
> 100
> 100
> 100
> 100
> 100
> 20
> 100
> 100
0.4
(
(
(
(
(
R)-PA-824
> 100
0.5
S)-CGI-17341
R)-CGI-17341
S)-Phenyloxazole
R)-Phenyloxazole
207.2 ± 70.8
123.2 ± 20.1
67.8 ± 37.5
2.4 ± 0.6
2.2 ± 0.2
1.7 ± 0.5
0.011 ± 0.002
0.020 ± 0.005
0.029 ± 0.011
0.25
12.5
0.8
0.2–0.4
12.5
83.6 ± 15.7
c
4.8 ± 0.5
c
0.061 ± 0.003
c
0.8
OPC-67683
Aza-PA-824
0.01
0.8
> 20
> 100
12.9 ± 3.4
1.6 ± 0.1
0.117 ± 0.015
> 100
a
b
c
H37Rv-5A1-DfbiC. H37Rv-14A1-DDdn. Not determined because of poor aqueous solubility of OPC-67683 (< 1 lM).
1
16
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M. Gurumurthy et al.
Biochemical characterization of Mtb Ddn
reinforcing the notion that Mtz killing of anaerobic
Mtb occurs via a mechanism that is independent of
F420 and Ddn. Aza-PA-824 was equally good as a sub-
(kcat ⁄ Km,app) for all four tested nitroimidazo-oxazole
analogs are largely driven by their high Km,app values.
This is possibly because of the absence of the trifluoro-
methoxy benzyl group which seems to be important
for Ddn enzyme activity [22].
strate as PA-824 for Ddn with a k ⁄ Km,app of
cat
)
1
)1
0
.12 lm Æmin (Fig. 2B and Table 1).
Previously, we have shown that Mtb Ddn mutants
Notably the maximal velocity of the (R) isomer was
nearly equal to that of PA-824 although the affinity
was significantly lower (Km,app 84 lm). Thus it seems
that Ddn displays stereospecificity with oxazole sub-
strates with larger lipophilic tails but the stereo prefer-
ence is the reverse of that observed with the oxazine
series.
were equally as sensitive as wild-type (WT) Mtb to the
nitroimidazo-oxazole analog CGI-17341 [19] suggesting
involvement of other Ddn homologs in its activation.
CGI-17341 is a racemic mixture that proved to be a
modest substrate for Ddn (data not shown). In order
to understand the enantiomeric specificity of nitroimi-
dazo-oxazoles, we synthesized the (R) and (S) stereoi-
somers of CGI-17341 (Data S1 ). Both (R) and (S)
forms had highly potent Mtb cellular activity against
WT H37Rv as well as against H37Rv-14A1-DDdn,
suggesting a Ddn independent mode of activation.
Nevertheless, activation was still F420-dependent as the
H37Rv-5A1-DfbiC strain was resistant to both the ste-
reoisomers (Table 1). These genetic studies suggest that
while PA-824 is activated exclusively by Ddn in Mtb,
activation of CGI-17341 is carried out by Ddn or
other Ddn homologs (Rv1261c, Rv1558 and Rv3178).
Although Ddn is not likely to be the only relevant
reductase for activation of CGI-17341, these com-
pounds nonetheless were Ddn substrates in vitro. Sub-
strate-dependent F420H2 oxidation was observed with
both the (R) and (S) isomers of CGI-17341 at about
half the maximal velocity achieved for PA-824 and
there was no significant rate difference between the
two enantiomers (Fig. 2B, Table 1).
Resistance to OPC-67683, the (R) isomer of a bicy-
clic 4-nitroimidazo-oxazole with a hydrophobic tail on
the oxazole ring, has also been mapped to Ddn [15].
We found OPC-67683 to be highly active against Mtb
H37Rv (MIC90 0.01 lm) whereas the Ddn mutant, as
expected, was resistant to this compound, confirming
that Ddn is the primary biological activator for the
compound (Table 1). OPC-67683 showed substrate-
dependent F420H2 oxidation by Ddn (with an initial
)
1
velocity of 63 nmÆmin with 5 lm OPC-67683). How-
ever, owing to solubility limitations with the com-
pound (< 1 lm in aqueous buffer), in vitro enzyme
kinetics of Ddn with OPC-67683 could not be evalu-
ated. Enzymatic formation of the des-nitro form of
OPC-67683 with Ddn was detected by LC-MS analysis
(Fig. S2, m⁄ z 490 compared with 535 for the parent)
consistent with the whole cell metabolite analysis
reported previously [15].
Guided by our earlier observations showing that
PA-824 resistant Ddn mutants were partially resistant
to nitroimidazo-oxazoles with longer hydrophobic sub-
stituents on the oxazole ring [19], we suspected that
substrates with a longer lipophilic tail would perhaps
stereoselectively interact with Ddn. To probe this
hypothesis further, we synthesized the (R) and (S) ste-
reoisomers of a nitroimidazo-oxazole with a bulkier
hydrophobic phenyl side-chain (Data S2). As expected,
the (R)-phenyloxazole was 15-fold more active in
whole cell activity against WT H37Rv than its corre-
sponding (S) enantiomer but similar activities of both
isomers were observed when minimal inhibitory con-
centrations (MICs) were determined with a Ddn
mutant (Table 1). Once again, this implicates the role
of other homologs of Ddn in Mtb such as Rv1261c,
Rv1558 and Rv3178 in the cellular activation of simple
nitroimidazo-oxazole analogs. When tested against
purified Ddn, both (R)- and (S)-phenyloxazole analogs
Kinetic mechanism of Ddn catalysis
The kinetic mechanism of Ddn was studied using preli-
minary two-substrate profile analyses (Fig. 2C,D). The
increase in the Vmax,app values with increasing concen-
trations of both PA-824 and F420H suggest that reac-
2
tion chemistry must occur through a ternary complex
of Ddn : F420H : PA-824 (Fig. 2E). However, further
2
analysis such as product inhibition studies are required
to determine precise binding constants and to distin-
guish between a random versus an ordered kinetic
mechanism. Detailed understanding of Ddn : F420H2 :
PA-824 catalysis would aid nitroimidazole optimi-
zation.
The kinetics of NO generation by Ddn
Previously, we used the Griess assay to study the
in vitro release of reactive nitrogen intermediates pro-
duced from the Ddn catalyzed PA-824 reaction [21]. In
this assay, a two-step diazotization reaction under
acidic conditions results in the formation of an
were poor substrates for Ddn with k ⁄ Km,app values
cat
)1
)
1
)1
)1
of 0.06 lm Æmin
(
and 0.03 lm Æmin
Table 1). Interestingly, the low specificity constants
respectively
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117

Biochemical characterization of Mtb Ddn
M. Gurumurthy et al.
azo-complex which can be monitored spectrophoto-
metrically. Although a reliable endpoint assay for Ddn
as reported, its low sensitivity (ꢀ 1 lm) is of concern
in the study of enzyme kinetics. Here, we employed an
NO analyzer based on chemiluminescence detection
with a sensitivity of ꢀ 1 nm (1000-fold better) to study
in vitro Ddn kinetics. Steady state kinetics of Ddn with
Ddn [21] and therefore did not show NO release
(Fig. 3A).
Ddn binding with (R)- and (S)-PA-824 and F420 by
fluorescence quenching
The N-terminal portion of Ddn contains a critical
tryptophan (Trp20) involved in binding the hydropho-
bic tail of PA-824. Fluorescence emission spectra of
1 lm Ddn in the presence of increasing concentrations
of PA-824 revealed that PA-824 efficiently quenched a
significant fraction of the intrinsic fluorescence of this
protein upon binding (Fig. 4A). The extent of ligand
binding to Ddn was determined by monitoring changes
in maximal fluorescence emission at 334 nm. At satu-
rating concentrations of PA-824, 69.6% of the intrinsic
Ddn fluorescence was quenched with no significant
change in the maximum emission wavelength. To fur-
ther explore the role of stereochemistry in determining
substrate binding, we also explored the inactive (R)-
PA-824 in this assay, which was incapable of turnover
by Ddn. Surprisingly (R)-PA-824 was able to bind to
the enzyme causing a reduction in intrinsic fluorescence
by 78% at saturating concentrations (Fig. 4B). How-
ever, under similar conditions, des-nitro PA-824 failed
to bind Ddn (Fig. 4C). The relative change in intrinsic
fluorescence 1 ) F ⁄F0 was plotted against the ligand
concentration to generate saturation isotherms
PA-824 and F420H in the presence of saturating con-
2
centrations of either substrate were investigated by
monitoring NO generation (Fig. 3A,B). The Km,app for
PA-824 was 8 lm and for F420H2 was 22 lm with a
)
1
)1
kcat ⁄ Km,app value of 0.01 lm Æmin . As expected, des-
nitro PA-824 and (R)-PA-824 were not substrates for
(Fig. 4D). In the absence of F420H , Ddn was able to
2
bind PA-824 and (R)-PA-824 with Kd values of
3.95 lm and 19.24 lm, respectively. Binding to the
1
oxidized or reduced F420 was evaluated in a similar
manner (Fig. 4E,F). Ddn displayed extremely tight
binding to oxidized F420 with a K of 0.30 lm in con-
d
trast to the reduced form that showed a Kd of 3 lm
(Fig. 4G). Moreover, binding of F420-2 to Ddn, moni-
tored by the same method, was comparable to that of
F420-5 from M. smegmatis suggesting that the extended
polyglutamate tail did not contribute significantly to
binding affinity (Fig. S3).
In order to obtain further understanding of how the
inactive (R) form of PA-824 still bound to Ddn despite
lack of turnover, we examined potential binding modes
of both forms to the co-crystal structure of Ddn with
F420 that we recently reported [21a]. The full-length
Ddn protein structure was not solved; however, the
high resolution crystal structure of a full-length related
enzyme from Nocardia (nfa33440; see Fig. S4 for
sequence alignment) as well as extensive mutagenesis
studies support a structural model of the active site.
We used this model to manually dock both isomers of
PA-824 to arrive at putative binding modes. In both
cases, a water molecule H-bonded to Ser72 was used as
Fig. 3. Monitoring Ddn kinetics using an NO analyzer. (A) Micha-
elis–Menten plots of the reaction of PA-824, des-nitro PA-824 and
(
R)-PA-824 with 1 lM Ddn at 100 lM F420
course of NO release profiles at varying PA-824 concentrations). (B)
Michaelis–Menten plot of the reaction of F420 with 1 lM Ddn at
0 lM PA-824. Each value represents the mean ± standard devia-
2
H (inset shows the time
H
2
5
tion of the initial velocity of formation of oxidized F420 from a linear
regression analysis.
1
18
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M. Gurumurthy et al.
Biochemical characterization of Mtb Ddn
Fig. 4. Binding of nitroimidazole and F420 substrates to Ddn evaluated by intrinsic tryptophan fluorescence quenching studies. Emission
spectra of 1 lM of Ddn protein in the presence of increasing concentrations of (A) PA-824, (B) (R)-PA-824, (C) des-nitro PA-824, (E) F420 and
(
F) F420
ligand. (D), (G) Saturation isotherms for Ddn binding with various ligands. K
ried out at least three independent times and one such representative profile is shown.
H
2
. Arrows indicate quenching of the fluorescence signal at the wavelength of maximum emission with increasing concentrations of
d
values are indicated in the graphs. The experiments were car-
a docking point for the nitro group. Figure 5A shows
that the C-3 of the nitroimidazole of the (S) isomer is
(Fig. 5B) and is skewed away from the C-5 of F420H2
and this makes the hydride transfer very unlikely. In
both cases, the aromatic tail portion is aligned well
with Trp16 for aromatic–aromatic interactions and this
is consistent with the fluorescence quenching experi-
ments. While not conclusive, this docking provides a
directly positioned above the C-5 of F420H (shown by
2
an asterisk in Fig. 1), and this allows a favorable
hydride transfer from the C-5 of F420H . However, the
C-3 of the (R) isomer docked in a flipped orientation
2
FEBS Journal 279 (2012) Journal compilation 113–125 ª 2011 FEBS. No claim to original US government works
119

Biochemical characterization of Mtb Ddn
M. Gurumurthy et al.
Fig. 5. Proposed docking modes of (S) and (R) isomers of PA-824. Binding modes of (A) catalytically active (S)-PA-824 and (B) catalytically
inactive (R)-PA-824 to F420 bound Nocardia Ddn homolog, nfa33440 (top panels). The respective bottom panels show the orientation of the
geometry optimized enantiomers (in yellow) docked into the active site of a Nocardia Ddn homolog complexed with F420 with a few impor-
tant residues in the binding pocket labeled. (A), Bottom panel: One of the oxygen atoms of NO
2
of (S)-PA-824 was placed onto the crystal
˚
water molecule to H-bond with Ser72 of the Nocardia homolog. In this orientation, the C-3 of the imidazole is 4.1 A away from the C-5 of
F420, and also properly positioned for the incoming hydride transfer in terms of the angle of C-5–(H)–C-3; note that the hydride (H) of C-5 is
not seen in the X-ray structure. Atoms represented by colors are as follows: green, carbon; blue, nitrogen; red, oxygen. (B), Bottom panel:
˚
The enantiomer (R)-PA-824 was docked in a flipped orientation resulting in the C-3 of the imidazole at a distance of 4.3 A away from the C-5
of F420 and an improper positioning for the incoming hydride transfer in terms of the angle of C-5–(H)–C-3.
plausible explanation for the observed stereoselectivity
of the (S) isomer of PA-824 over the (R) isomer by
Ddn as well as their comparable binding affinity to
Ddn.
metal chelating reagent EDTA suggests that the
enzyme activity is metal ion independent. Ddn is a 151
amino acid protein with only one cysteine residue at
position 149 (Fig. S4), implying that only intermolecu-
lar disulfide bonds may exist, if at all. DTT, up to
2
mm, had no effect on the activity of Ddn suggesting
Discussion
the lack of any structurally or functionally important
intermolecular disulfide bonds. Addition of Triton
X-100 improved the enzyme activity significantly; the
presence of a surfactant possibly stabilizes Ddn, there-
fore contributing to improvement in activity.
The possibility of emergence of drug resistance war-
rants further study and optimization of second genera-
tion nitroimidazoles as anti-tuberculars [22,23,26,27].
The kinetic properties of analogs of this class as sub-
strates for Ddn are important determinants of their
activity against the whole organism. Therefore a more
detailed biochemical characterization of the reaction is
important to support a deeper understanding of the
structure–activity relationship in this series and to
guide optimization efforts. Ddn enzyme activity was
unaffected by the inclusion of monovalent or divalent
cations. Also, the lack of any effect upon addition of a
Consistent with recently reported F420 bound crystal
structure of the core enzyme lacking the N-terminus
[21a], Ddn was highly specific to F420H and was not
2
able to utilize several other cofactors which exhibit sim-
ilar reduction potentials (nicotinamide based cofactors)
or resemble F420 structurally (flavins) [25]. Ddn could
utilize reduced F420H2 as efficiently as the reduced
F420-5 from M. smegmatis. Therefore, although the po-
1
20
FEBS Journal 279 (2012) Journal compilation 113–125 ª 2011 FEBS. No claim to original US government works

M. Gurumurthy et al.
Biochemical characterization of Mtb Ddn
lyglutamate chain of F420 seems to be important for
Ddn activity, the number of glutamate residues (rang-
ing from 2 to 6) did not seem to contribute to nitroimi-
dazole conversion.
family involved in bioremediation of 2,4,6-trinitrotolu-
ene and at least one of these enzymes has been
shown to be F420-dependent [28,29]. The deazaflavin
cofactor F420 is unique in that under normal condi-
tions it is an obligate two electron or hydride ion
donor [20]. However, the precise role of the low
The kinetic constant kcat ⁄ Km,app for the Ddn enzy-
)
1
)1
matic reaction with PA-824 was 0.16 lm Æmin . Ddn
was not able to metabolize either the (R) stereoisomer
or the des-nitro derivative of PA-824. Tryptophan
quenching studies indicated that Ddn was able to bind
PA-824 and (R)-PA-824 with similar affinity. These
results demonstrate that the physical binding of Ddn to
PA-824 is not affected by the stereochemistry of the
compound; however, the complete loss of activity of
the enzyme with the (R) stereoisomer suggest that
redox potential of the 5-deaza-cofactor (F420H ) in
2
the mechanism of bicyclic nitroimidazole reduction is
yet to be understood.
Here we also describe a highly sensitive and robust
method for examining in vitro Ddn enzyme kinetics
based on NO detection using an NO analyzer. The
anaerobicidal activity of bicyclic 4-nitroimidazoles cor-
related with the formation of des-nitro species and
release of reactive nitrogen intermediates [21].
Although the two nitroimidazole clinical candidates
for TB in phase II trials, PA-824 and OPC-67683, have
modest anaerobic activity, they have been optimized
only for aerobic whole cell activity. An assay that
determines the release of NO will therefore facilitate
target based selection of PA-824 analogs that have
improved anaerobic activity. The kcat ⁄ Km,app of
PA-824 determined from the NO assay was
(
R)-PA-824 may bind to Ddn non-productively and
interfere with the catalysis of the enzyme probably due
to the incorrect positioning of C-3 of the nitroimidazole
ring to C-5 of the F420H2 from which the hydride
transfer takes place. Molecular docking studies of
(
(
R)-PA-824 with Ddn strengthen this hypothesis
Fig. 5). Although we have some preliminary evidence
that (R)-PA-824 is a competitive inhibitor of Ddn med-
iated reduction of (S)-PA-824 (data not shown), the
very poor solubility of this compound precluded full
characterization of the inhibition. These results, how-
ever, suggest that clinical development of a racemic
mixture would not be advisable. The des-nitro deriva-
tive showed negligible binding to Ddn supporting the
lack of enzyme activity with des-nitro PA-824 as a sub-
strate, suggesting a critical role for the nitro group in
binding. Binding studies of Ddn with the F420 indicated
higher affinity for F420 (Kd 0.3 lm) than F420H2 (Kd
)1
)1
0.013 lm Æmin which is 10 times lower than what is
observed in the F420 oxidation assay. The efficiency of
NO generation or detection seems to be reduced possi-
bly due to quenching of NO in solution under the con-
ditions tested or the non-enzymatic reaction between
NO and F420H [30]. Consequently the utility of this
2
assay needs to be reconfirmed after careful analysis of
detection of various reactive nitrogen intermediates
and reaction conditions. However, in the presence of
3
lm). This binding of oxidized F420 in the active site
saturating concentrations of F420H (100 lm) and with
2
could prevent the entry of a reduced F420 into the
active site possibly explaining the low turnover of
the enzyme. However, the low binding constant for the
product might change in the presence of the nitroimi-
dazole substrate, which requires further investigation.
4 lm PA-824, 1.12 lm NO was produced in 2 h along
with 7.2 lm of oxidized F420 (implying 155 nm of NO
produced per micromole of F420 oxidized). Thus in
Mtb cells release of micromolar concentrations of
intracellular NO by Ddn would have a significant
effect on cell viability.
The F420H -dependent reduction of PA-824 by Ddn
2
produces three stable metabolites. As proposed earlier
21], the first hydride transfer from the C-5 of F420H2
The physiological role of Ddn is currently unknown.
Sequence homologs primarily exist in actinobacteria;
however, there is little information on their potential
cellular functions. Ddn was first experimentally
detected in a study that identified membrane proteins
in Mtb suggesting that Ddn might be a membrane
associated protein [31]. Rv1261c, Rv1558 and Rv3178
are the three homologs of Ddn in Mtb that have other
orthologs in other mycobacterial species. Ddn and its
homologs form a class of previously uncharacterized
[
to the C-3 of PA-824 results in a nitronic acid interme-
diate of PA-824 from which all of the three metabo-
lites arise. The des-nitro derivative of PA-824 (a major
metabolite) is formed by elimination reaction with the
release of nitrous acid, which in turn disproportionates
to release NO and other reactive nitrogen species. Fur-
ther chemical transformations and F420H2 mediated
reduction result in the formation of exocyclic ketone
and exocyclic imine. The chemistry involving reduction
F420H -dependent nitroreductases with no identified
2
of the C=C bond by F420H through the formation of
2
physiological substrate. A recent bioinformatics study
that carried out phylogenetic profiling of 1451 bacterial
and archaeal genomes based on F420 biosynthesis
a Meisenheimer complex is well precedented in
enzymes of the bacterial old yellow enzyme (OYE)
FEBS Journal 279 (2012) Journal compilation 113–125 ª 2011 FEBS. No claim to original US government works
121

Biochemical characterization of Mtb Ddn
M. Gurumurthy et al.
nominated three dominant families, one of which was
the Ddn family [32]. The study indicates that the Ddn
family is restricted to F420 producing bacteria. Another
250 mL column chromatography. F420 was further purified
using reverse phase (Source RPC 30; GE Healthcare) chro-
matography (acetonitrile gradient, 400 nm). A final yield of
1
1
.18 lmolÆL) culture was obtained which was comparable
study that identified F420H -dependent reductases in
2
)
1
with the 1.43 lmolÆL reported in the literature [36]. Puri-
fied F420 was characterized by LC-ESI MS and fluorescence
emission scanning. As expected, F420 showed an emission
maximum at 470 nm when excited at 400 nm. Each of the
peaks in the LC profile corresponded to one of the F420
analogs (Fig. S1A,B). As reported earlier [24], F420 with
four to six glutamate residues accounted for the majority of
the F420 analogs in the purified sample.
the degradation of aflatoxins showed that a Ddn
homolog in M. smegmatis, Msmeg_5998, utilized
F420H in the reduction of the unsaturated ester moiety
2
of aflatoxins, thereby activating them for spontaneous
hydrolysis [33,34]. Both the degradation of aflatoxin
and reduction of PA-824 by Ddn seem to share a com-
mon mechanism in which hydride is transferred from
the low redox potential deazaflavin to the electron defi-
cient ring systems of the substrates.
The biochemical characterization of Ddn described
here with PA-824 and other analogs, and the methods
developed, will prove useful in future attempts to char-
acterize additional members of the Ddn enzyme family.
In addition, since Ddn appears to be capable of rigor-
ous structure-based specificity, characterizing this pro-
tein for its binding to the various nitroimidazole
analogs will prove to be beneficial to optimize a
backup clinical candidate for PA-824.
Cloning, expression and purification of
recombinant Ddn protein
The coding sequence for Ddn (Rv3547) was amplified by
PCR (Pfx polymerase; Invitrogen) from H37Rv genomic
DNA using forward (5¢-caggatcccgaaatcaccgccgcggtt-3¢)
and reverse (5¢-tagcggccgctcagggttcgaaaccacga-3¢) primers.
PCR amplified fragments were cloned into BamHI and
NotI sites of an entry vector of a Gateway expression sys-
tem [37]; further the gene was sub-cloned into various desti-
nation vectors. A construct (pMBPKan5-Ddn) coding for
N-terminal MBP-His6-tagged Ddn was transformed into
BL21 (DE3) Tuner cells and protein expression was
induced with 0.1 mm IPTG for 20 h at 18 ꢀC. Soluble
recombinant MBP-His6-Ddn was purified on a nickel affin-
ity binding column and the fusion protein was cleaved by
PreScission protease (GE Healthcare). The digested protein
was subjected to ion exchange chromatography (Resource
S; GE Healthcare) and the elutant was passed through a
second nickel affinity binding column to remove any
remaining tagged protein and non-removed tags. The puri-
fied protein was stored at )80 ꢀC in a buffer containing
Experimental procedures
Bacteria and culture conditions
E. coli DH5a strain used for cloning and E. coli BL21
(DE3) Tuner strain used for protein expression were grown
in Luria broth as per standard protocol. MIC90 determina-
tion and culture conditions for Mtb H37Rv WT, H37Rv-
5A1-DfbiC and H37Rv-14A1-DDdn have been described
earlier [19].
2
0 mm Tris ⁄ HCl pH 7.5, 100 mm NaCl, 10% glycerol,
mm DTT. The purified protein was a soluble aggregate
Drugs and chemicals
1
and had a calculated size of ꢀ 17.5 kDa on SDS ⁄ PAGE
Mtz and isopropyl thio-b-d-galactoside (IPTG) were obtained
from Sigma-Aldrich (Singapore, Singapore). (S)-PA-824,
(
Fig. S1C) which corresponded to the predicted molecular
weight. Protein concentration of 3.03 lgÆlL) was obtained
as determined by the standard Bradford assay [38] and the
final yield of the purified protein was about 30 mgÆL) of
culture.
1
(R)-PA-824 and Aza-PA-824 [23] and OPC-67683 [35] were
synthesized as described. Synthesis of (R) and (S) isomers of
both CGI-17341 and a phenyloxazole derivative are described
in Data S1 and Data S2. All compounds were dissolved in
1
9
0% DMSO as 50 mm stocks.
Evaluation of Ddn enzyme activity
F420 purification from M. smegmatis
Enzymatic activity was determined spectrophotometrically
by monitoring F420H2 oxidation at A400 nm (isosbestic
point for oxidized F420) [21]. F420H2 was prepared as
described previously from F420 by FGD1 enzyme in the
presence of glucose-6-phosphate as the substrate [21]. F
concentration was measured at A400 (e = 25.7 mm)1Æcm)1).
Typically, the assay mixture to determine enzyme activity
contained 50 lm PA-824, 100 lm F420H2 and 1 lm Ddn
in a final volume of 100 lL unless specified otherwise.
2
420 was purified from M. smegmatis mc 155 cells using a
F
modified version of a protocol described earlier [36]. Briefly,
M. smegmatis was grown in large batches in modified
strullu-romand (MSR) medium in a fermentor; cells were
harvested, resuspended in water and lysed by three rounds
of autoclaving (10 min at 121 ꢀC). The lysed cell extract
was filtered and passed through an HiQ (Biorad) ion
exchange 250 mL column followed by Florisil (Sigma)
4
20
1
22
FEBS Journal 279 (2012) Journal compilation 113–125 ª 2011 FEBS. No claim to original US government works

M. Gurumurthy et al.
Biochemical characterization of Mtb Ddn
Control reactions without the enzyme and without PA-824
were included for each set of experiments. To determine
Apparent kinetic constants Vmax,app, Km,app and kcat ⁄ Km,app
for each substrate were determined by fitting the initial rate
of NO release as a function of substrate concentration
using nonlinear regression fitted to the Michaelis–Menten
equation.
the pH optimum for Ddn activity, F420
monitored at room temperature at pH ranging from 6.0 to
.0 (Mes ⁄ OH pH 6.0–6.5, Tris ⁄ HCl pH 7.0–7.5,
2
H oxidation was
8
HEPES ⁄ NaOH pH 8.0–8.5). For temperature optima, the
reaction was carried out in an optimal buffer (referred to
as Ddn buffer: 200 mm Tris ⁄ HCl, pH 8.0, with 0.01% Tri-
ton X-100) at varying temperatures between 20 and 50 ꢀC.
The effect of detergent (Triton X-100), monovalent cations
Ddn binding studies to PA-824, F420 and F420H2
Fluorescence emission spectra of Ddn (excitation at
280 nm) in a 100 lL volume with or without ligand were
recorded between 310 and 420 nm using a Tecan Infinite
M1000 plate reader. Ddn, 1 lm, was incubated with
increasing ligand concentrations for 30 min before fluores-
cence measurements. The extent of ligand binding to Ddn
was determined by monitoring changes in maximal fluores-
+
+
2+
2+
[
Na , K (0–300 mm)], divalent cations [Mg , Mn
(0–20 mm)], DMSO (0–10%), EDTA (0–20 mm) and
reducing agent DTT (0–2 mm) on enzyme activity was
evaluated by including them in the reaction mixture. To
2
determine specificity of Ddn for F420H , this component in
the assay reaction was replaced with NADH and
NADPH. For Ddn kinetics experiments, the initial veloci-
cence emission at 334 nm. The dissociation constant K
d
value ([ligand] at which 50% of the protein’s intrinsic tryp-
tophan fluorescence is quenched) was calculated by fitting
the relative change in intrinsic fluorescence at 334 nm
2
ties of F420H oxidation were plotted against the substrate
concentration (PA-824 and its analogs) and analyzed using
nonlinear regression to the Michaelis–Menten equation
using graphpad prism 5 (GraphPad Software Inc., La
Jolla, CA, USA). When nanomolar concentrations of Ddn
were used in order to achieve steady state kinetics at lower
concentrations of nitroimidazole substrates, detection of
(1 ) F ⁄ F ) of Ddn versus ligand concentration to a one-site
0
binding (hyperbolic model) using nonlinear regression,
where F0 is the intrinsic intensity of fluorescence of 1 lm
Ddn alone and F is the fluorescence at a given concentra-
tion of ligand; corrections were made to subtract ligand-
associated fluorescence.
F
420
H
2
oxidation was not feasible since the rate of Ddn
catalyzed F420 oxidation was comparable to the sponta-
neous re-oxidation of in the absence of the
enzyme. Therefore 1 lm of Ddn was used in all reactions
and apparent kinetic constants
cat ⁄ Km,app for any given reaction were determined from
the plotted data.
H
2
F
420 2
H
Modeling of PA-824 at the binding pocket
V
max,app
, Km,app and
Putative binding modes of both stereoisomers of PA-824 in
the binding pocket of Ddn were investigated using high res-
olution co-crystal structures of F420 with Ddn and with a
homolog from Nocardia (nfa33440) [21a]. The pseudo-
equatorial conformer of (S)-PA-824 was constructed by
replacing the methyl of 7R-methyl-PA-824 [39] with a
hydrogen, and then energy minimized in the gaseous phase
with the density functional theory at the level of B3LYP ⁄ 6-
31G* [40]. The geometry of (R)-PA-824 was obtained by
inversion of (S)-PA-824 coordinates. Docking of both (S)-
PA-824 and (R)-PA-824 to the binding pocket was done
manually with Quanta 2008 (Accelrys).
k
Ddn kinetics studies via NO release assay
NO release was directly monitored using the Sievers NO
analyzer 280i. The NO analyzer consists of an ozone gener-
ator, a chemiluminescent reaction chamber and a red-sensi-
tive photomultiplier tube (PMT). In an aqueous reaction
buffer, reactive nitrogen species react with dissolved O
form nitrite, which is reduced to NO by sodium iodide
1%) in acetic acid. The chemiluminescence assay is based
on a gas phase reaction between NO and ozone to generate
electronically excited nitrogen dioxide (NO *) which emits
2
to
(
2
Acknowledgements
light in the red and near-infrared and is detected by a ther-
moelectrically cooled, red-sensitive PMT. The reaction
chamber containing glacial acetic acid–NaI with 100 lL of
This work was funded, in part, by a grant from the
Bill and Melinda Gates Foundation and the Well-
come Trust through the Grand Challenges in Global
Health Program 11 to NITD through Imperial Col-
lege London; by NITD and also in part by the intra-
mural research program of NIAID (to CEB) and
through the Center for Information Technology (to
YSL). We thank Lacy Daniels for F420-2. The quan-
tum chemical study utilized PC ⁄ LINUX clusters at
the Center for Molecular Modeling of the NIH
1
· antifoaming agent was purged with nitrogen continu-
ously. The reaction chamber was connected to the PMT;
standard 50 lL solutions of sodium nitrite (1 nm to 1 lm)
were injected and a calibration curve was generated with
nitrite concentration as a function of the PMT output
(mV). Varying concentrations of nitroimidazole substrates
or F420
H
2
were used with 1 lm of Ddn in a Ddn buffer.
NO release profiles were monitored over a 10-min period
injecting 50 lL of the reaction mixture at each time point.
(http://cit.nih.gov).
FEBS Journal 279 (2012) Journal compilation 113–125 ª 2011 FEBS. No claim to original US government works
123

Biochemical characterization of Mtb Ddn
M. Gurumurthy et al.
the treatment of tuberculosis. Nature 405,
62–966.
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Fig. S1. (A) LC MS chromatograms at 280 nm (top)
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MS. (C) SDS⁄ PAGE showing the expression and puri-
fication of recombinant Ddn: M, marker; 1, uncleaved
MBP-His6-Ddn; 2, cleaved Ddn; 3, 4, 5, flow through,
wash and eluate respectively from ion exchange col-
umn. MBP-His-Ddn and cleaved Ddn proteins are
denoted by arrows. (D), (E) Effect of pH and tempera-
ture on the activity of recombinant Ddn enzyme. (F),
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Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be reorganized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
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