THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 48, pp.
37860 –37871, November 26, 2010
Phosphorylation of Enoyl-Acyl Carrier Protein Reductase
InhA Impacts Mycobacterial Growth and Survival*
Received for publication, May 10, 2010, and in revised form, September 10, 2010 Published, JBC Papers in Press, September 23, 2010, DOI 10.1074/jbc.M110.143131
Shazia Khan‡1, Sathya Narayanan Nagarajan‡, Amit Parikh‡2, Sharmishtha Samantaray‡, Albel Singh§,
Devanand Kumar¶, Rajendra P. Roy‡, Apoorva Bhatt§3, and Vinay Kumar Nandicoori‡4
From the ‡National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067 India, the §School of Biosciences, University of
Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom, and the ¶Department of Microbiology, University of Delhi South
Campus, New Delhi 110021, India
InhA, the primary target for the first line anti-tuberculosis
drug isoniazid, is a key enzyme of the fatty-acid synthase II sys-
tem involved in mycolic acid biosynthesis in Mycobacterium
tuberculosis. In this study, we show that InhA is a substrate for
mycobacterial serine/threonine protein kinases. Using a novel
approach to validate phosphorylation of a substrate by multiple
kinases in a surrogate host (Escherichia coli), we have demon-
strated efficient phosphorylation of InhA by PknA, PknB, and
PknH, and to a lower extent by PknF. Additionally, the sites
targeted by PknA/PknB have been identified and shown to be
predominantly located at the C terminus of InhA. Results
demonstrate in vivo phosphorylation of InhA in mycobacte-
ria and validate Thr-266 as one of the key sites of phosphor-
ylation. Significantly, our studies reveal that the phosphor-
ylation of InhA by kinases modulates its biochemical activity,
with phosphorylation resulting in decreased enzymatic activ-
ity. Co-expression of kinase and InhA alters the growth
dynamics of Mycobacterium smegmatis, suggesting that InhA
phosphorylation in vivo is an important event in regulating its
activity. An InhA-T266E mutant, which mimics constitutive
phosphorylation, is unable to rescue an M. smegmatis condi-
tional inhA gene replacement mutant, emphasizing the criti-
cal role of Thr-266 in mediating post-translational regulation
of InhA activity. The involvement of various serine/threonine
kinases in modulating the activity of a number of enzymes of
the mycolic acid synthesis pathway, including InhA, accentu-
ates the intricacies of mycobacterial signaling networks in
parallel with the changing environment.
The characteristic nature of the cell envelope of Mycobacte-
rium tuberculosis is linked to its pathogenicity. The thick layer
of lipids on the outer surface of mycobacteria is protective in
nature, and mycolic acids comprise the bulk of this layer.
Mycolic acids also constitute structural components of the cell
wall and envelope (1, 2). Mycobacteria are unique in having two
* This work was supported in part by the Department of Biotechnology, India
(to V. K. N. and R. P. R.).
1
Senior Research Fellow of the Council of Scientific and Industrial Research,
India.
2
Department of Biotechnology postdoctoral fellow.
3
Supported by a career development award from the Medical Research
Council, United Kingdom.
4
To whom correspondence should be addressed. Tel.: 91-11-26703789; Fax:
91-11-26742125; E-mail: vinaykn@nii.res.in.
37860 JOURNAL OF BIOLOGICAL CHEMISTRY
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
fatty-acid synthase (FAS)5 systems, FAS-I and FAS-II, and both
of these pathways are involved in the synthesis of mycolic acids.
Eukaryotic-like FAS-I is a single multidomain enzyme, whereas
FAS-II includes discrete monofunctional enzymes that carry
out the various sequential steps involved in the process of syn-
thesis (3). The FAS-I system is responsible for the de novo fatty
acid biosynthesis (4), producing C20- and C26-S-enzyme deriv-
atives that are converted to the respective coenzyme A (CoA)
forms and then released. The C20 fatty acid is the probable
starting point for the FAS-II system (2), and the C26 fatty acid is
incorporated as the ␣-branch of mycolic acid (5).
FabH (␤-ketoacyl-ACP synthase III) forms the link between
the two FAS systems by condensing the long chain acyl-CoA
products (produced by FAS-I) and malonyl-ACP (produced
from malonyl-CoA by the action of FabD (malonyl-CoA:ACP
transacylase) (6)). The ␤-ketoacyl-S-ACP product (7) thus
formed is subjected to keto-reduction, dehydration, and enoyl
reduction consecutively, reactions catalyzed by MabA (␤-keto-
acyl-ACP reductase), ␤-hydroxyl-ACP dehydratase complex
(Rv0636 ⫹ Rv0635, 0637), and InhA (enoyl-ACP reductase),
respectively (2, 8). The ACP-bound acyl chain thus becomes
two carbon units longer than the starting chain and now under-
goes repetitive cycles of reduction, with the exception that the
ketosynthases KasA or KasB substitute for FabH. InhA, the
2-trans-enoyl-ACP reductase of the FAS-II system (9), was first
identified as the gene encoding a target of the drugs isoniazid
and ethionamide, following the characterization of genetic
variants that conferred resistance to these drugs (10). Biochem-
ical analysis of InhA revealed that InhA catalyzes NADH-spe-
cific reduction of 2-trans-enoyl-ACP with preference for long
chain substrates (C12–C24) (11). InhA is also essential for the
survival of mycobacteria; inactivation of InhA affects mycobac-
teria acutely, causing accumulation of saturated fatty acids, the
end products of FAS-I (12), “blebbing” of the cell wall, and con-
sequent cell death by lysis (12, 13).
Protein phosphorylation is a principal mechanism by which
extracellular signals are transduced into cellular responses. M.
tuberculosis genome encodes for 11 eukaryote-like serine/thre-
onine protein kinases (STPKs) (14 –17). The M. tuberculosis
STPKs affect key mycobacterial processes. Signal transduction
events mediated by PknA and PknB play an important role in
5
The abbreviations used are: FAS, fatty-acid synthase; MBP, maltose-binding
protein; Ni-NTA, nickel-nitrilotriacetic acid; ACP, acyl carrier protein; STPK,
serine/threonine protein kinase.
VOLUME 285 • NUMBER 48 • NOVEMBER 26, 2010
 Regulation of Mycobacterial Enoyl-ACP Reductase InhA
determining cell shape, morphology, and possibly cell division
(18); PknG (19 –21) and PknH (22) influence M. tuberculosis
virulence, adaptation, and growth within the host; and PknF
affects cell division, growth rate, morphology, and glucose
transport (23). The mycobacterial FAS-II multienzyme com-
plex has been identified to be a target of M. tuberculosis STPKs,
with substrates, including the malonyl-CoA:ACP transacylase
FabD and the ␤-ketoacyl-ACP synthases KasA and KasB. These
proteins are phosphorylated by various kinases in vitro; how-
ever, the phosphorylation is found to be most efficient with
PknA. Interestingly, although phosphorylation has a negative
impact on the activity of KasA, KasB activity is enhanced upon
phosphorylation (24, 25). Recently, MabA, the ␤-ketoacyl-ACP
reductase, has been demonstrated to be negatively regulated
following phosphorylation (26). FabH (␤-ketoacyl-ACP syn-
thase III) has also been shown to be a substrate of several
kinases, particularly PknA and PknF. Phosphorylation of FabH
has an inhibitory effect on enzyme activity (27).
In this study, we have identified InhA to be a substrate of
multiple mycobacterial STPKs. InhA phosphorylation medi-
ated by the STPKs has been validated in vivo, using both Esch-
erichia coli as a surrogate host as well as in Mycobacterium
smegmatis. The putative phosphorylation sites have been iden-
tified. We demonstrate that the phosphorylation status of InhA
is critical to the function and/or activity of InhA and alters the
growth profile of mycobacteria. Most importantly, results from
studies carried out with an inhA conditional gene replacement
strain emphasize the crucial role of the phosphorylation status
of InhA in mediating mycobacterial survival.
EXPERIMENTAL PROCEDURES
Reagents and Radioisotopes—Restriction/modification en-
zymes were obtained from New England Biolabs. Cloning and
expression vectors pENTR/Directional TOPO cloning kit
(Invitrogen), pMAL-c2X (New England Biolabs), pGEX-4T-2
(GE Healthcare), pQE2 (Qiagen), and pET-Duet1 (Novagen)
were purchased from respective sources. pMV261-apra and
pJAM2 (E. coli-Mycobacterium shuttle vectors) were kind gifts
from Dr. William Jacobs and Dr. Tanya Parish, respectively.
[␥-32P]ATP (6000 Ci/mmol) was purchased from PerkinElmer
Life Sciences. Oligonucleotide primers and analytical grade
chemicals were purchased from Sigma.
Generation of Plasmid Constructs—M. tuberculosis pknA and
pknB (full length) were PCR-amplified, respectively, from BAC
clone Rv13 (a kind gift from Prof. Stewart Cole, Pasteur Insti-
tute, France) using gene-specific primers and Phusion DNA
polymerase (New England Biolabs). pknG (full length) and
kinase domains, including the region up to transmembrane
domains of pknD, pknE, pknF, pknH, pknI, pknJ, and pknL, were
amplified from BAC clones Rv313 (pknG), Rv103 (pknD),
Rv302 (pknE and pknF), Rv407 (pknH), Rv209 (pknI), Rv412
(pknJ), and Rv269 (pknL), respectively. pknA, pknD, pknE, pknF,
pknG, and pknI amplicons were digested with EcoRI and
HindIII and cloned into corresponding sites on pMAL-c2X
(New England Biolabs). pknB, pknH, and pknL amplicons were
digested with BamHI and HindIII and cloned into correspond-
ing sites on pMAL-c2X vector. pknJ amplicon was digested with
XbaI and HindIII and cloned into corresponding sites on
NOVEMBER 26, 2010 • VOLUME 285 • NUMBER 48
pMAL-c2X vector. pMAL-PknK (full length) and pGEX-PknB
were generated as described previously (28, 29). pJAM-PknA
and pJAM-PknB were created by cloning the PCR amplicon
obtained using the specific primers into BamHI/XbaI sites of
pJAM2 vector (30).
inhA gene was amplified from H37Rv BAC clone Rv58 using
gene-specific primers and cloned into pENTR/Directional
TOPO cloning vector (Invitrogen). inhA was further subcloned
into NdeI/NotI sites in pQE2 vector (Qiagen). The point
mutants used in this study were generated by overlapping PCR
mutagenesis using appropriate mutagenesis primers (details
are available upon request). inhA gene was amplified using spe-
cific primers, and amplicon was cloned into BamHI/XbaI sites
in MCS of pJAM2 to generate pJAM-InhA. To generate
pMV261apra-InhA wild type and mutant constructs, the
respective wild type and mutant pQE2-InhA constructs were
used as templates. Primers used for the amplification were such
that amplicon obtained included sequence for His tag at the N
terminus. Amplicons obtained were cloned into PvuII/HindIII
sites of pMV261-apra vector.
For the co-expression studies, the dual expression vector
pET-Duet1 (Novagen) was used. The different MBP-tagged
kinase constructs were digested with HindIII, and the over-
hangs generated were filled in using Klenow polymerase fol-
lowed by heat inactivation and digestion with NdeI. These frag-
ments were subcloned into MCS2 of pET-Duet1 at NdeI/
EcoRV sites. InhA was cloned into the NotI site in the first MCS
of pET-Duet1 vector. Strains and constructs used in this study
are described in Table 1.
Expression and Purification of Recombinant Proteins—Plas-
mids overexpressing PknA, PknB, and InhA (wild type and
mutants), or plasmids co-expressing a kinase and InhA, were
transformed into E. coli BL21 (DE3) Codon Plus cells (Strat-
agene). Cultures were induced with 0.1 mM isopropyl 1-thio-␤-
D-galactopyranoside and further grown for 12–16 h at 18 °C.
Cells were harvested and lysed by sonication in lysis buffer (20
mM Tris-HCl (pH 7.5), 200 mM NaCl, and 10 mM ␤-mercapto-
ethanol for MBP fusion proteins or phosphate-buffered saline
(pH 7.4), 10 mM ␤-mercaptoethanol for His-tagged proteins).
The cell lysates containing MBP or His fusion proteins were
nutated with equilibrated amylose resin (New England Biolabs)
or Ni-NTA-agarose (GE Healthcare) affinity resins, respec-
tively. His-tagged proteins were eluted with lysis buffer con-
taining 200 –250 mM imidazole. For the elution of MBP-tagged
proteins, 10 –20 mM maltose was used. GST-PknB was purified
as described earlier (29). Peak fractions were dialyzed against
dialysis buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, and 20%
glycerol or PBS containing 20% glycerol).
In Vitro Kinase Assay, Phosphoamino Acid Analysis, and
Phosphopeptide Mapping—In vitro kinase assays were per-
formed by incubating 1–5 pmol of PknA or PknB and 25–30
pmol of InhA (WT or mutants) as described earlier (29). Phos-
phoamino acid analysis and peptide mapping were carried out
as described previously (29, 31).
Preparation of M. smegmatis Cell Lysates—Electrocompe-
tent cells of M. smegmatis mc2155 were prepared, and 500 ng of
plasmid DNA was electroporated as described previously (32).
Large scale cultures were inoculated with 1% of primary culture
JOURNAL OF BIOLOGICAL CHEMISTRY 37861
Regulation of Mycobacterial Enoyl-ACP Reductase InhA
TABLE 1
Strains and constructs used in this study
and grown with shaking for 36 – 40 h. Cultures were harvested
and lysed using a bead beater (BioSpec Products), and the lysate
was clarified by centrifugation at 13,200 rpm for 50 min at 4 °C.
The lysate was incubated with Ni-NTA affinity beads to allow
for pulldown of His-tagged protein, and beads were washed
with PBS and resuspended in 2⫻ SDS sample buffer.
Two-dimensional Gel Electrophoresis—20 ␮g of InhA puri-
fied from E. coli strains co-expressing InhA and MBP/MBP-
PknA/MBP-PknB were precipitated with 5 volumes of cold
acetone. Precipitated proteins were resuspended in 300 ␮l of
two-dimensional sample buffer (33). Aliquots of the samples
were analyzed on SDS-PAGE to normalize the loading for the
two-dimensional gels. Equal amounts (⬃15 ␮g) of the samples
were resolved by two-dimensional gel electrophoresis as
described earlier (33). Gels were either silver-stained or trans-
ferred onto nitrocellulose membranes and probed with anti-
InhA antibodies raised in mice. For evaluating the InhA and
37862 JOURNAL OF BIOLOGICAL CHEMISTRY
InhA-T266A mutant expressed in M. smegmatis by two-di-
mensional gels, ⬃7.5 mg of lysates prepared from M. smegmatis
strains harboring pMV261-InhA or pMV261-InhA-T266A or
pJAM-InhA were used for pulldowns using Ni-NTA resin to
isolate His-InhA, His-InhA-T266A, and InhA-His, respec-
tively. Ni-NTA beads were resuspended in PBS containing 2%
SDS to elute bound proteins. Proteins thus eluted were ace-
tone-precipitated and resuspended in 300 ␮l of two-dimen-
sional sample buffer. Aliquots of the samples were analyzed on
SDS-PAGE, and ⬃5 ␮g of His-InhA or His-InhA-T266A or
InhA-His was resolved by two-dimensional gels followed by
Western blotting using anti-InhA antibodies. To examine
endogenous InhA, 300 ␮g of M. smegmatis mc2155 whole cell
lysates were precipitated with acetone followed by two-dimen-
sional gels and Western blotting using anti-InhA antibodies.
Enoyl-CoA Reductase Assay and Determination of Kinetic
Parameters—2-trans-Octenoyl-CoA was prepared from trans-
2-octenoic acid by the mixed anhydride method as reported
previously (34). The sample was purified by reverse phase-
HPLC (C18 250 ⫻ 4.60-mm column) using an acetonitrile/
water/TFA (trifluoroacetic acid) solvent system (gradient,
4 –72% B in 50 min; A, 0.1% TFA; B, acetonitrile containing
0.1% TFA; flow rate, 1 ml/min). The chromatogram was mon-
itored at 210 and 265 nm. Fractions containing 2-trans-oc-
tenoyl-CoA were pooled, lyophilized, and stored at ⫺20 °C till
use. The chemical identity of the compound was confirmed by
electrospray mass spectrometry.
InhA activity was assayed essentially as described previously
(11), with minor modifications. Briefly, the reactions were car-
ried out in a total volume of 50 ␮l in 30 mM PIPES buffer (pH
6.8) containing 500 ␮M 2-trans-octenoyl-CoA, 150 ␮M NADH,
and 5–50 pmol of InhA enzyme (InhA or p-InhA, respectively).
Time course assays were carried out, monitoring oxidation of
NADH by measuring absorbance at 340 nm for 15 min, at
1-min intervals (GE Ultrospec 2100pro). NAD formed was cal-
culated as ((A340 at 0 min ⫺ A340 at time t (1–15 min)/A340 at 0
min) ⫻ (NADH ␮M/pmol of InhA) and plotted as a function of
time.
To determine the kinetic constants with respect to 2-trans-
octenoyl-CoA, reactions were carried out in a 50-␮l volume of
30 mM PIPES containing 150 ␮M NADH, 15 or 30 pmol of InhA
or p-InhA, respectively, and varying concentrations of 2-trans-
ocetenoyl-CoA. The reaction was performed for 15 min, and
the absorbance was measured at 340 nm. NAD formed was
calculated as ((initial A340 ⫺ final A340/initial A340) ⫻ (NADH
␮M/min/mg enzyme)). Km and Vmax values were determined by
nonlinear regression analyses carried out with GraphPad Prism
software. Data from two independent experiments were plotted,
and results are presented as average values ⫾ S.D. Similarly, kinetic
constants were determined for NADH in 50-␮l reactions contain-
ing 500 ␮M 2-trans-octenoyl-CoA, 15 or 30 pmol of InhA or
p-InhA, respectively, and varying concentrations of NADH.
Growth Curve Analysis—M. smegmatis mc2155 strains, con-
taining either pMV261apra or pMV261apra-InhA, were elec-
troporated with pJAM-PknA (acetamidase-inducible). To carry
out analysis of PknA, InhA, or PknA ⫹ InhA overexpression in
mycobacteria, each strain was grown in 7H9-ADC without
inducer to the exponential phase and re-inoculated into fresh
VOLUME 285 • NUMBER 48 • NOVEMBER 26, 2010
 Regulation of Mycobacterial Enoyl-ACP Reductase InhA
medium (in the presence and absence of 0.2% acetamide) such
that A600 was 0.02 for the secondary culture at time 0. Samples
were withdrawn at different time points for A600 measurement.
Studies of the growth pattern of InhA mutants were similarly
carried out. M. smegmatis mc2155 strains electroporated with
pMV261 vector only, pMV261-InhA, pMV261-InhA T266A,
and pMV261-InhA T266E were used to overexpress the respec-
tive proteins. For analyzing effects of PknA co-expression with
various InhA mutants, M. smegmatis mc2155 was electropo-
rated with the integration-proficient construct pST-HI-acet-
PknA. The resulting strain contains a single copy of pknA under
the control of the acetamidase promoter. pMV261 vector,
pMV261-InhA, pMV261-InhA T266A, and pMV261-InhA-
T266E were electroporated into this strain. The resulting
strains were grown in LB/Tween 80 without inducer to expo-
nential phase and re-inoculated into fresh medium (in the pres-
ence and absence of 0.5% acetamide) such that A600 was 0.02 for
the secondary culture at time 0. Samples were withdrawn at
different time points for A600 measurement.
Assessment of in Vivo Functionality of InhA Phosphorylation
Mutants—pMV261-InhA, pMV261-InhA-T266A, and pMV261-
InhA-T266E were introduced into the M. smegmatis inhA con-
ditional mutant strain mc24751 (13) by electroporation to
generate the transformed strains, mc24751/pMV261-inhA,
mc24751/pMV261-inhA-T266A, and mc24751/pMV261-inhA-
T266E. The ability of the transforming inhA allele to rescue
growth defects of the conditional mutant was assessed by grow-
ing the transformants in the presence or absence of acetamide
(0.2% in tryptic soy broth) as described earlier (13).
CD Spectroscopic Analyses—Protein samples were prepared
in PBS (pH 7.4). CD spectra were recorded on Jasco J-710 spec-
tropolarimeter. Data were collected at 25 °C using 2-mm path
length cell from 250 to 190 nm in 1-nm steps, at a scanning
speed of 200 nm/min. Spectra presented are the averages of 20
scans. The results are reported as mean residue ellipticity
expressed as degrees cm2 dmol⫺1.
Structural Modeling—Three-dimensional models of the
InhA T266A and T266E mutants were created using PyMOL
software (35). The Protein Data Bank code 1BVR of the pub-
lished M. tuberculosis InhA structure (36) was used as the tem-
plate for rendering these in silico structures.
RESULTS
InhA Is a Novel Target of M. tuberculosis STPKs—Kang et al.
(18) have demonstrated that PknA and PknB show preferential
phosphorylation of Thr residues that precede a Gln residue.
Interestingly, several substrates of these kinases identified to
date conform to this motif, including KasB of the FAS-II system
(24). The presence of a TQ motif in InhA, and considering that
several other enzymes of the FAS-II pathway were found to be
substrates for PknA and/or PknB, we set out to explore whether
InhA could also be a target for mycobacterial STPKs. Toward
this, inhA, pknA, and pknB from M. tuberculosis were cloned
and the respective proteins overexpressed in and purified from
E. coli (Fig. 1A). In vitro kinase reactions carried out with
recombinant InhA and PknA or PknB demonstrated that InhA
was phosphorylated by both kinases (Fig. 1B). The InhA thus
phosphorylated was subjected to phosphoamino acid and pep-
NOVEMBER 26, 2010 • VOLUME 285 • NUMBER 48
FIGURE 1. InhA is an in vitro substrate of PknA and PknB. A, E. coli BL21
(DE3) codon plus cells were transformed with pQE2-InhA, pMAL-PknA, and
pGEX-PknB, respectively, and the corresponding His-InhA, MBP-PknA, and
GST-PknB proteins were purified as described under “Experimental Proce-
dures.” Bands corresponding to the purified proteins are indicated. B, in vitro
kinase assays carried out using MBP-PknA (5 pmol) or GST-PknB (1 pmol) with
InhA (30 pmol) in the presence of [␥-32P]ATP. Bands corresponding to auto-
phosphorylated PknA or PknB and phosphorylated InhA are indicated by
arrowheads. C, phosphoamino acid analysis of in vitro phosphorylated InhA.
The panel shows the autoradiogram and the positions of pSer, pThr, and pTyr
standards on the TLC plates are indicated (p ⫽ phosphorylated). D, two-di-
mensional peptide map of in vitro phosphorylated InhA autoradiogram is
shown.
tide mapping analyses. It was observed that InhA is phosphor-
ylated specifically on threonine residues by PknA (Fig. 1C) and
PknB (data not shown). The peptide map obtained was similar
for InhA phosphorylated by PknA (Fig. 1D) and PknB (data not
shown) and shows the presence of multiple spots, indicating
that InhA is phosphorylated on multiple sites by these kinases
in vitro.
InhA Is Phosphorylated by Multiple STPKs—Many substrates
identified thus far are phosphorylated by multiple M. tubercu-
losis kinases. To date, reports demonstrating phosphorylation
of substrate by multiple kinases have been carried out in vitro
using purified substrates and kinases. To investigate phosphor-
ylation of InhA by M. tuberculosis serine/threonine kinases, we
developed a convenient approach, utilizing E. coli as a surrogate
host. pDuet-1 vector has two multiple cloning sites (MCS1 and
MCS2) with independent T7 promoters, thus enabling co-ex-
pression of two proteins. We cloned either the full-length
kinase (PknA, PknB, PknG, and PknK) or the kinase domains,
including the region up to the transmembrane domain (kinase
domain) (PknD, PknE, PknF, PknH, PknI, PknJ and PknL) in
pMAL-c2x vector as described under “Experimental Proce-
dures.” All 11 MBP-tagged kinases and the MBP tag were inde-
pendently subcloned into the MCS2 of pDuet1 vector (Fig. 2A).
As a first step, we determined the expression status of MBP and
the MBP-tagged kinases. It is apparent from the results (Fig. 2B)
that MBP and the MBP-tagged kinase expressed robustly.
Restriction sites that are unique in MCS1 and can be used for
substrate cloning are shown in Fig. 2A. Upon cloning, the sub-
strate of interest into MCS1 of pDuet-MBP/MBP kinase con-
structs, one can express both the substrate and the kinase from
JOURNAL OF BIOLOGICAL CHEMISTRY 37863
Regulation of Mycobacterial Enoyl-ACP Reductase InhA
PknB, respectively, by means of the
specific pDuet constructs. Phos-
phorylation status of InhA was
determined by probing the His pull-
downs with phosphothreonine anti-
bodies (Fig. 3A). It was evident that
both PknA and PknB phosphory-
lated InhA efficiently in vivo. To
ascertain whether InhA is a target of
any of the other STPKs, we per-
formed an analogous experiment
with the other kinases, with InhA
co-expressed with MBP and MBP-
PknB as negative and positive refer-
ence points, respectively (Fig. 3B).
Our results showed that in addition
FIGURE 2. pDuet-1 constructs designed for co-expression of MBP/MBP kinase and substrate. A, schematic to PknA and PknB, PknH and to an
representation of the MCS region of pDuet-MBP/MBP kinase constructs cloned in MCS2. Unique sites that can extent PknF also phosphorylated
be used for cloning test substrate in MCS1 are shown. B, expression profiles of the various pDuet-MBP/MBP
kinase constructs. Respective constructs were transformed in BL21 (DE3) BL21 codon plus cells, and corre- InhA. Together, these results indi-
sponding cultures were induced. Lysates were probed with anti-MBP antibodies (New England Biolabs). KD cate that InhA is a target substrate
refers to kinase domains, including the region up to transmembrane region.
of multiple kinases that probably
phosphorylate the protein on
more than one threonine residue.
InhA Is Phosphorylated in the C-terminal Region—Primary
sequence analysis of InhA showed the presence of only one
“TQ” motif. The peptide map of InhA phosphorylated by
PknA/PknB, however, indicated that there are multiple phos-
phorylation sites, with threonines specifically being targeted
(Fig. 1, C and D). Analysis of the InhA primary sequence
revealed presence of 13 threonine residues. We mutagenized all
13 potential phosphorylation sites individually or in combina-
tion (Thr-253 and Thr-254), from threonine to alanine resi-
dues, to determine which sites would modify the phospho-
peptide maps. Nine of the twelve mutants had the same
phosphopeptide pattern as the wild type InhA (data not
shown). The peptide maps for the other three mutants, namely
T79A, T266A, and T253A,T254A, were distinct from that of
the wild type InhA. Spots that either disappeared or showed
substantial decrease in intensity as compared with the wild type
map are numbered from 1 to 7 (Fig. 4A). Because all the TLC
plates were subjected to autoradiography for the same period of
time, we correlated the disappearance and/or decreased inten-
sity with the phosphorylation on a particular site. Peptide map
of InhA-T79A showed absence of spot 1, indicating phosphor-
FIGURE 3. InhA is targeted by multiple STPKs of M. tuberculosis. A, pDuet ylation of InhA on the Thr-79 residue. Mutation of Thr-266
constructs with MBP, MBP-pknA, or MBP-pknB cloned in MCS2 with or without resulted in disappearance of spot 2 and decreased intensities of
InhA cloned in MCS1 were transformed in BL21 (DE3) codon plus cells. Lysates
and His pulldowns were probed with anti-Thr(P) (pThr) (Cell Signaling), anti- spots 4, 6, and 7. In the case of T253A,T254A mutant, we
His (BD Biosciences), and anti-MBP (New England Biolabs) antibodies. observed a decrease in the intensities of spots 3, 5, and 6. Maps
B, pDuet-1 co-expression constructs containing MBP or kinases in MCS2 and
InhA in MCS1 were subjected to Western blot analysis as above. KD refers to of T253A and T254A single mutants were similar to the peptide
kinase domains, including the region up to transmembrane region. Top panel, map of InhA-T253A,T254A double mutant (data not shown).
Thr(P) blot; middle panel, His blot performed on His pulldowns; lower panel, Theoretical tryptic digestion of InhA revealed that the C-ter-
MBP blot on lysates to show the expression of kinases.
minal tryptic fragment has four threonine residues (Thr-241,
Thr-253, Thr-254, and Thr-266). Mutation of one or the other
a single construct in E. coli BL21 codon plus strain, thus provid- threonines may have influence on the in vitro phosphorylation
ing an in vivo tool to assess phosphorylation of the substrate by of neighboring threonine residues in the peptide. This may lead
corresponding kinase. to further changes in the intensities of spots we detect on TLC.
To validate the in vitro phosphorylation of InhA by PknA and The complexity of the maps owing to a combination of multiple
PknB, InhA was co-expressed with MBP, MBP-PknA, or MBP- phosphorylation sites, more than one threonine residue on a

37864 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 48 • NOVEMBER 26, 2010
 Regulation of Mycobacterial Enoyl-ACP Reductase InhA
gel, as negative charge on the phos-
phate group results in a shift toward
the acidic end of the IPG strip. This
property of a differential migration
pattern of phosphorylated proteins
was used to determine whether
the various phosphorylated forms
of InhA could be distinguished
by two-dimensional gel electro-
phoresis. InhA was purified from
E. coli co-expressing InhA and
MBP, MBP-PknA, or MBP-PknB.
These proteins were resolved in
two dimensions, and the protein
spots were visualized by silver stain-
ing and Western blotting with
InhA-specific antibodies (Fig. 5A).
Compared to InhA co-expressed
with MBP, InhA co-expressed with
either PknA or PknB showed addi-
tional spots, demonstrating phos-
phorylation of InhA by these
kinases (Fig. 5A). The spot intensi-
ties as calculated by ImageJ software
indicated that ⬃50% of InhA pro-
tein was phosphorylated by PknA
FIGURE 4. InhA is phosphorylated at its C terminus. A, in vitro phosphorylated InhA mutants were digested or PknB, respectively, under these
with trypsin, and the resulting phosphopeptides were mapped by two-dimensional resolution on thin layer conditions.
chromatography. The spots undergoing a change in relative intensity in the InhA-mutant peptide maps com- Next, to ascertain whether InhA
pared with the wild type InhA map are numbered 1–7. B, comparative kinase assay of InhA wild type and
various single and combination threonine point mutants. 5 pmol of MBP-PknA and 30 pmol of InhA or InhA is indeed phosphorylated in myco-
mutants were used for the assays. Upper panel, autoradiogram; lower panel, Coomassie-stained protein bands bacteria, we transformed M. smeg-
of the InhA mutants. C, 4 ␮g of His-InhA or His-InhA- T253A,T254A,T266A (T253,254,266A) purified from E. coli
co-expressing MBP or MBP-PknB were resolved, transferred, and probed with anti-phosphothreonine antibod-
matis with plasmid encoding M.
ies. Similarly, 500 ng of the proteins were resolved and probed using anti-His antibodies. Lane 1, His-InhA tuberculosis inhA (pMV261-InhA),
co-expressed with MBP; lane 2, His-InhA co-expressed with MBP-PknB; lane 3, InhA-T253,254,266A mutant purified the recombinant InhA
co-expressed with MBP-PknB.
bearing N-terminal His tag, and
analyzed the protein by two-dimen-
tryptic peptide and/or the partial tryptic digestions did not per- sional gel electrophoresis. Western blot analysis of the two-
mit complete assignment of all the spots. dimensional gels showed the presence of multiple spots, sug-
Next, generated combination threonine site mutants of gesting phosphorylation of InhA in M. smegmatis (Fig. 5B,
InhA, and carried out comparative kinase assays with wild upper panel). Results in Fig. 4 demonstrated that InhA was
type and various InhA mutants (Fig. 4B). We observed a mainly phosphorylated on Thr-253, Thr-254, and Thr-266.
decrease in the extent of phosphorylation for most of the We generated pMV261-InhA-T266A, pMV261-InhA-T253A,
mutants when contrasted with the wild type InhA. Notably, T254A, and pMV261-InhA-TriT, plasmid-borne versions of
the combination mutant in which threonine residues at posi- the mutants, and transformed them into M. smegmatis. How-
tions 253, 254, and 266 are mutated to alanine concurrently ever, we could only detect expression of InhA and InhA-T266A
(TriT: T253A,T254A,T266A mutant) showed significantly mutant; the other mutants failed to express despite the
decreased phosphorylation (Fig. 4B). To substantiate these sequence analysis of the clones showing no discrepancy. We
findings, we purified InhA from E. coli strains co-expressing resolved the His-InhA-T266A mutant isolated from M. smeg-
either the MBP or MBP-PknB with InhA wild type or the TriT matis by two-dimensional gel electrophoresis. It is clear from
mutant and analyzed the phosphorylation status of InhA (Fig. our results (Fig. 5B, middle panel) that mutating the Thr-266
4C). It is apparent from the results that the triple mutant was residue resulted in disappearance of spots. These results
negligibly phosphorylated by the kinase as compared with the strongly suggest that Thr-266 is a bona fide phosphorylation
wild type InhA. Taken together, our results suggest that Thr-79 site of InhA. Furthermore, because the phosphorylation of
is a minor phosphorylation site, whereas Thr-253, Thr-254, and InhA occurs mainly at the C terminus, we reasoned that addi-
Thr-266, located in the C-terminal region, are major targets of tion of a hexa-His tag at the C-terminal protein may interfere
PknA/PknB-mediated phosphorylation events. with the phosphorylation. Hence, we generated a pJAM-InhA
InhA Is Phosphorylated in Vivo—Phosphorylation is known construct with C-terminal His tag. Interestingly, when InhA-
to alter the pattern of protein migration in a two-dimensional His protein purified from M. smegmatis transformed with

NOVEMBER 26, 2010 • VOLUME 285 • NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 37865
Regulation of Mycobacterial Enoyl-ACP Reductase InhA
FIGURE 5. InhA is phosphorylated in vivo, and Thr-266 is a major target
site. A, His-InhA protein purified from E. coli following co-expression with
MBP, MBP-PknA, or MBP-PknB was precipitated with 5 volumes of cold ace-
tone. Approximately 15 ␮g of precipitated proteins were resolved on a 13-cm
pH 4 –7 linear gradient IPG strips (GE Healthcare) for 25,000 V-h. IPG strips
were resolved in the second dimension on 10% SDS-polyacrylamide gels and
were either visualized by silver staining or transferred to nitrocellulose mem-
brane and subjected to Western blotting with anti-InhA antibodies raised in
mice. B, two-dimensional gels of InhA protein isolated from M. smegmatis
overexpressing InhA from pMV261-InhA, pMV261-InhA-T266A, or pJAM-
InhA, respectively. Approximately 5 ␮g of the respective proteins were
resolved as described. Western blots of the resolved proteins probed with
anti-InhA antibodies are shown. C, 300 ␮g of M. smegmatis mc2155 whole
cell lysate was precipitated with acetone, followed by resolution using
two-dimensional gels as above, followed by Western blotting using anti-
InhA antibodies.
pJAM-InhA was resolved by two-dimensional gels, Fig. 5B, bot-
tom panel, we observed a pattern very similar to that observed
for InhA-T266A. These results are in agreement with our ear-
lier data that showed phosphorylation of InhA in the C-termi-
nal region (Fig. 4). To verify that InhA is phosphorylated at
endogenous expression levels, M. smegmatis whole cell lysate
was resolved by two-dimensional gel electrophoresis, trans-
ferred to nitrocellulose membrane, and probed with anti-InhA
antibodies. We observed multiple spots (Fig. 5C), suggesting
that endogenous InhA is phosphorylated in mycobacteria. The
number of spots detected was less than those observed for the
His-InhA, most likely due to significantly lower amounts of
the InhA protein present in the total lysates.
Phosphorylation of InhA Affects Its Biochemical Activity—
InhA from M. tuberculosis has been shown to catalyze NADH-
specific reduction of 2-trans-enoyl-CoA with preference for
long chain substrates (C12–C24), with C16 –C20 substrates
being most efficiently reduced. A chain length of C8 was the
minimum required to be a substrate for M. tuberculosis InhA
(11). We synthesized 2-trans-octenoyl-CoA, as reported previ-
ously (34), for use in an in vitro enzyme assay. The experimental
mass of the 2-trans-octenoyl-CoA was in accord with its calcu-
lated mass (891.29 Da), and the HPLC trace of the final product
showed presence of a single peak (data not shown).
InhA co-expressed with MBP or MBP-PknB in E. coli was
purified to represent unphosphorylated and phosphorylated
forms of the protein, InhA and p-InhA, respectively. Phosphor-
ylation of InhA by PknB was confirmed by Western blotting
with anti-phosphothreonine antibodies (Fig. 6A). Activity of
37866 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 6. Phosphorylation of InhA affects its biochemical activity. A, InhA
proteins used for enoyl-CoA reductase assay, visualized by Coomassie stain-
ing (top panel), His blot (middle panel), and phospho-blot (lower panel). Lane 1,
InhA purified from E. coli co-expressing MBP tag; lane 2, InhA purified from
E. coli co-expressing MBP-PknB that represents phosphorylated protein
(p-InhA) (p ⫽ phosphorylated). B, graphical representation of time course
assays of InhA (F) and p-InhA (E), carried out as detailed under “Experimental
Procedures.” The y axis corresponds to NAD formed (␮M/pmol of InhA) at each
time point, and the x axis signifies time (min). Average values of three inde-
pendent experiments are presented; the error bars represent the standard
error.
TABLE 2
Kinetic parameters of M. tuberculosis InhA and p-InhA
Experimental conditions and data analysis are described under “Experimental Pro-
cedures.” Data from two independent experiments are presented as average val-
ues ⫾ S.D.
InhA p-InhA
2-trans-Octenoyl-CoA Km (␮M) 528 ⫾ 16.7 511 ⫾ 13
NADH Km (␮M) 19.1 ⫾ 5.4 57.8 ⫾ 5.2
Vmax (␮M product/min/mg enzyme) 15.3 ⫾ 2.4 3.4 ⫾ 0.8
phosphorylated (p-InhA) and nonphosphorylated InhA was
determined by measuring NADH oxidation over time. The
NAD formed at each time point was calculated and plotted as a
function of time for both the enzyme sets (Fig. 6B). We
observed ⬃5-fold reduction in the activity for p-InhA com-
pared with nonphosphorylated InhA, suggesting negative mod-
ulation of InhA enzymatic activity upon phosphorylation. Fur-
thermore, we determined the kinetic parameters for InhA and
p-InhA for both the substrates 2-trans-octenoyl-CoA and
NADH (Table 2). Specific activity of InhA was found to be
⬃5-fold higher compared with p-InhA in agreement with our
previous results (Fig. 6B). However, if the stoichiometry of the
phosphorylation was taken into consideration (⬃50% utilizing
pDuet co-expression system), the difference in the specific
activity would be ⬃10-fold. We observed similar Km values for
2-trans-octenoyl-CoA for both the enzymes, although InhA
seems to have higher affinity for NADH compared with p-InhA
(Table 2).
Phosphorylation of InhA Impacts Growth of Mycobacterium—
Loss of InhA activity leads to slowing of mycobacterial growth
and subsequent lysis of cells (12, 13). Given the relatively lower
activity of phosphorylated InhA as compared with the non-
phosphorylated form in our enzyme assays (Fig. 6A), we sought
to determine the effects of phosphorylation of InhA on the
growth pattern of mycobacteria. For this purpose, M. smegma-
tis was transformed with plasmids that expressed either InhA
(pMV261-InhA) or PknA (pJAM-PknA) or both. An acet-
amide-inducible promoter in pJAM-PknA allowed controllable
expression of PknA. Western blot analysis demonstrated inde-
pendent as well as co-expression of InhA and PknA (Fig. 7A).
VOLUME 285 • NUMBER 48 • NOVEMBER 26, 2010
 Regulation of Mycobacterial Enoyl-ACP Reductase InhA
Similar efforts to co-express PknB
and InhA in M. smegmatis were
unsuccessful, although PknB ex-
pression by itself was quite stable.
InhA overexpression alone had no
discernible effects on the growth of
M. smegmatis (Fig. 7B). However,
we observed that growth of M.
smegmatis expressing PknA was
compromised even in the absence of
inducer, likely due to basal expres-
sion from the acetamidase pro-
moter. This was not surprising, as
overexpression of PknA has been
shown to affect the growth of the
organism (18). While our results
were compounded by the fact that
PknA expression per se had a dele-
terious effect on mycobacterial
growth (Fig. 7B) and (18), we did
observe a further quantifiable de-
crease in the growth of cultures
expressing both InhA and PknA,
compared with the cultures ex-
pressing only PknA (Fig. 7B). This
suggested that the PknA-mediated
phosphorylation of InhA may play a
role in modulating the growth pat-
tern of the bacterium.
To investigate this hypothesis
further, we created an InhA-T266E
mutant. The glutamate residue
would mimic a phosphorylated
threonine residue, and hence this
mutant was expected to function
like InhA constitutively phosphor-
ylated at the Thr-266 position. We
studied the growth pattern of M.
smegmatis strains transformed with
plasmids for overexpression of
FIGURE 7. Co-expression of PknA and InhA leads to diminished growth in mycobacteria. A, expression InhA, InhA-T266A, and InhA-
profile of M. smegmatis strains overexpressing pknA and/or inhA from pJAM-PknA and pMV261-InhA con-
structs, respectively. His pulldowns were subjected to Western blotting with anti-PknA (raised in rabbit) or T266E, alongside a control strain.
anti-InhA antibodies (raised in mice). B, growth pattern analysis of M. smegmatis strains overexpressing pknA Surprisingly, the expression levels
and/or inhA. InhA expression was constitutive, whereas PknA expression was under the control of acetamide- of these mutants varied drastically
inducible promoter. Growth of strains containing vector control (pJAM2) only (F), vector control (pJAM2) and
InhA (E), PknA only (
), PknA in presence of acetamide (ƒ), PknA and InhA (f), and PknA and InhA in presence (Fig. 7C). We observed that M.
of acetamide (䡺) was monitored over a period of 48 h, every 3 h. A600 is plotted as a function of time. Average smegmatis expressing the T266E
absorbance values at each time point from three independent experiments are plotted; the error bars repre-
sent the standard deviation of each data set. C, expression profile of M. smegmatis strains overexpressing InhA, mutant exhibited a slower growth
InhA-T266A, or InhA-T266E from respective pMV261-InhA constructs. His pulldowns subjected to Western rate compared with either InhA or
blotting with anti-InhA antibodies. D, growth curves of M. smegmatis strains overexpressing InhA, InhA-T266A, InhA-T266A (Fig. 7D). Further-
and InhA-T266E. Growth of strains containing vector control (pMV261 apra) only (F), InhA (E), InhA-T266A (
),
and InhA-T266E (ƒ) were monitored over a 40-h period at an interval of every 3 h. A600 is plotted as a function more, we attempted to co-express
of time as above. E, expression profile of M. smegmatis strains overexpressing pknA and/or inhA mutants from the InhA, InhA-T266A, and InhA-
pST-HI-acet-PknA (integrated single copy of PknA) and pMV261-InhA/pMV261-InhA-T266A/pMV261-InhA-
T266E constructs, respectively. His pulldowns were subjected to Western blotting with anti-PknA or anti-InhA
T266E mutants, respectively, with
antibodies. F, growth pattern analysis of M. smegmatis strains overexpressing pknA and/or inhA/inhA T266A. PknA. For this purpose, the M.
InhA expression was constitutive, whereas PknA expression was under the control of acetamide-inducible smegmatis strain containing the sin-
promoter. Growth of strains containing PknA only (F), PknA in presence of acetamide (E) and InhA (
), PknA
and InhA (ƒ), and PknA and InhA in presence of acetamide (f), InhA-T266A (䡺), PknA and InhA-T266A (⽧), and gle integrated acetamidase-induci-
PknA and InhA-T266A in presence of acetamide (〫) was monitored over a period of 40 h. A600 is plotted as a ble copy of PknA was used as parent
function of time as above. strain (PknA merodiploid). Unlike
ectopically expressing pJAM-PknA,

NOVEMBER 26, 2010 • VOLUME 285 • NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 37867
Regulation of Mycobacterial Enoyl-ACP Reductase InhA
the single integrated copy of PknA serves to minimize the det-
rimental effects of PknA overexpression on growth. PknA
merodiploid strain was electroporated with pMV261,
pMV261-InhA, pMV261-InhA-T266A, and pMV261-InhA-
T266E, respectively. Despite repeated attempts, we could not
co-express InhA-T266E along with PknA. Analysis of growth
patterns (Fig. 7F) showed that co-expression of InhA-T266A
with PknA partially alleviates the diminution in growth com-
pared with PknA-InhA co-expression (Fig. 7F). Inability of
InhA-T266A mutant co-expression alongside PknA to com-
pletely revert the diminution of growth to only PknA overex-
pression levels could be due to the possibility that sites other
than Thr-266, such as Thr-253 and Thr-254, may be still be
targeted by PknA. Our results in Fig. 6B showed that phosphor-
ylation of InhA led to decreased catalytic activity. Taken
together, these results suggest that overexpressed PknA may be
hyperphosphorylating InhA, which may result in decreased
activity of InhA, and hence significantly compromised growth.
Phosphorylated State of InhA Is Functionally Inactive in Vivo—
To examine the in vivo relevance of the observed differences,
we tested the ability of the above described mutant alleles of
inhA to rescue an M. smegmatis conditional inhA gene replace-
ment mutant from death by lysis. The M. smegmatis mc24751
strain contains a deletion in the native inhA gene and has a
recombinant, acetamide-inducible copy of inhA integrated into
the chromosome (13). This strain grows normally in medium
containing acetamide, whereas growth in medium devoid of
acetamide results in loss of mycolic acid biosynthesis and sub-
sequent cell lysis. The mutant alleles of inhA can thus be func-
tionally analyzed in vivo by testing the ability of this strain
transformed with plasmid-borne copies of the mutant alleles to
survive (no lysis) in the medium devoid of acetamide. Strains of
M. smegmatis mc24751 transformed with pMV261-InhA-
T266A or pMV261-InhA-T266E were cultured in medium with
or without acetamide as described previously (13). Strains con-
taining vector only (pMV261apra) or native inhA (pMV261-
InhA) were used as controls. As expected, although mc24751/
pMV261apra lysed in medium without acetamide, mc24751
transformed with pMV261-InhA was able to grow equally well
in medium with or without acetamide (Fig. 8). The strain
mc24751/pMV261-InhA-T266A also grew normally in me-
dium devoid of acetamide. On the other hand, mc24751/
pMV261-inhA-T266E showed lysis when cultured in acet-
amide-free medium, thus indicating that the InhA-T266E
mutant was unable to function efficiently in vivo. In addition,
viable counts observed for vector control were ⬃10-fold higher
compared with mc24751/pMV261-inhA-T266E, thus suggest-
ing that InhA-T266E may be behaving like a dominant negative
form. These findings stress the importance of kinase-mediated
phosphorylation of InhA.
Gross Conformation of InhA Is Unaffected by Mutations at
Thr-266 Residue—It is clear from our data that InhA-T266E
mutant could not functionally complement WT-InhA. We
investigated whether this could be a consequence of conforma-
tional aberrations caused by the InhA-T266E mutation. To ver-
ify that the overall conformational features of wild type are
maintained in the mutant protein, we purified InhA and InhA-
T266E mutants and analyzed them by CD spectroscopy (Fig. 9,
37868 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 8. Assessment of the role of InhA phosphorylation in vivo. The top
panel shows cultures of mc24751 strains transformed with different plasmids
following 24 h of growth in TSB with or without acetamide. The lower panel
corresponds to viable counts obtained from each tube, where 10-fold serial
dilutions were spotted (5 ␮l) on TSB-agar plates containing acetamide.
A and B). The spectra of the two proteins in Fig. 9B are very
similar suggesting that the T266E mutation does not perturb
the gross folding of InhA. Additionally, in silico modeling of the
three-dimensional structure of the InhA-T266E mutant using
the published structure of M. tuberculosis WT InhA as a tem-
plate revealed no apparent changes in the structure of the
mutated protein or the substrate binding pocket (Fig. 9C). InhA
is known to form homodimers. Hence, it is possible that T266E
mutant may act as the dominant negative enzyme by dimeriz-
ing with wild type InhA. Combined with our kinetic data, we
speculate that subtle conformational changes, if any, due to
increase in charge because of phosphorylation might also mod-
ulate NADH binding and catalysis.
DISCUSSION
M. tuberculosis kinases have a plethora of substrates (16),
including several key enzymes of the FAS system (24, 26, 27, 37).
As the substrates identified to date in the FAS pathway are
proficiently phosphorylated by PknA, we scrutinized the pri-
mary sequence of all the enzymes in the FAS pathway for the
presence of the consensus “TQ” motif. Our analysis revealed
that KasB, which was shown to be a substrate of these kinases
(24), and InhA harbor this motif. The possibility of InhA also
being a part of the signaling network of these kinases thus
became a subject of interest to us. Our data illustrate robust
phosphorylation of InhA by PknA and PknB, in agreement with
the prediction of this enzyme being a target substrate (Fig. 1).
InhA is an important component of the FAS-II pathway, the
loss of which results in retarded growth and subsequent cell
lysis (12, 13). Our in vitro InhA activity assay results suggested
that phosphorylation of InhA results in decreased enzymatic
activity (Fig. 6). Furthermore, in line with these results, we
found that the strain expressing InhA-T266E has a delayed
growth phenotype compared with the strain expressing the
wild type form of the protein (Fig. 7). Most importantly,
the InhA-T266E mutant failed to functionally complement the
inhA conditional gene replacement mutant (Fig. 8) (13), imply-
ing phosphorylation-dependent modulation of InhA function.
The fact that a number of enzymes involved mycolic acid bio-
VOLUME 285 • NUMBER 48 • NOVEMBER 26, 2010
 Regulation of Mycobacterial Enoyl-ACP Reductase InhA
substrates using all 11 M. tuberculo-
sis STPKs (Fig. 2). This involves use
of a dual expression vector pDuet-1.
We have generated a series of 12
pDuet constructs harboring MBP
kinase/MBP tag in MCS2; the puta-
tive substrate can be cloned in
MCS1 (Fig. 2). The resulting plas-
mids are ideal for co-expression of
the kinase-substrate pair, respec-
tively, in the surrogate host E. coli.
As the phosphorylation event takes
place in vivo in the surrogate host,
results obtained are free of the prob-
lems associated with in vitro reac-
tions. This methodology provides
an excellent in vivo tool, which can
be used widely to assess phosphory-
lation of potential substrate by all 11
kinases of M. tuberculosis. Using
this method, we have been able to
demonstrate that PknA, PknB, and
PknH efficiently target InhA, al-
though PknF also targets it but to a
lower extent (Fig. 3).
Identifying phosphorylation sites
on a target substrate is a challenging
task. We used a combination of pep-
tide maps with mutant substrates
and comparative kinase assays to
determine the phosphorylation sites
on InhA. A detailed scrutiny indi-
cated that major sites of phosphor-
ylation were present in the same
tryptic peptide of InhA. Our inabil-
ity to account for all the spots
observed could be due to the pres-
ence of multiple phosphorylation
sites on the same peptide (Fig. 4). In
conjunction with kinase assays, we
identified Thr-253, Thr-254, and
FIGURE 9. Gross conformation of InhA is unaffected by mutations at Thr-266. A, 1 ␮g of purified InhA and Thr-266 to be the major targets for
InhA-T266E were resolved on 10% SDS-PAGE and visualized by Coomassie staining. B, CD spectra of InhA PknA/PknB-mediated phosphoryl-
(green) and InhA-T266E (blue) are presented as a measure of mean residue ellipticity (MRE). C, in silico modeling
of InhA mutants. The top panel represents superimposed ribbon diagrams and electrostatic surface potential ation and validated the same (Fig. 4).
maps (residue 266 is shown within a dotted circle). The bottom panel shows the opposite face of the protein Two-dimensional gels have been
where the substrate binding pocket can be visualized. used to confirm in vivo phosphory-
lation status of KasA, KasB, and
synthesis pathway, including InhA, are regulated by serine/ FabH (24, 27). In agreement with our in vitro results, two-di-
threonine protein kinase-mediated phosphorylation empha- mensional gel electrophoresis analysis of InhA co-expressed
sizes the crucial role of the phosphorylation in mediating with PknA or PknB in E. coli demonstrated phosphorylation on
mycobacterial survival. multiple sites (Fig. 5). InhA isolated from M. smegmatis strain
A complete understanding of phosphorylation events in a harboring pMV261-InhA plasmid appears to be effectively phos-
global context entails determining whether a substrate is part of phorylated as seen by two-dimensional gel analysis. We also vali-
a larger network involving multiple kinases. Thus far, in vitro dated Thr-266 to be a major phosphorylation site in vivo in myco-
kinase assays with purified M. tuberculosis kinases and the sub- bacteria by means of the same approach (Fig. 5). Furthermore, we
strate in question have been employed by various groups to observed multiple spots when M. smegmatis lysates were probed
address this vital issue (24, 26, 27, 38, 39). We have devised an with anti-InhA antibodies (Fig. 5), suggesting phosphorylation of
expedient method to assess the potential candidacy of protein InhA under endogenous expression conditions.

NOVEMBER 26, 2010 • VOLUME 285 • NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 37869
Regulation of Mycobacterial Enoyl-ACP Reductase InhA
Phosphorylation-dephosphorylation events are a mode of
regulating cellular functions. STPK-mediated phosphorylation
events in M. tuberculosis are no exception to this paradigm.
Several illustrations have been well documented in the litera-
ture (37). Phosphorylation events of the transcriptional regula-
tor EmbR affect its ATPase activity and binding with DNA (40).
Interactions of anti-anti-␴ factor homolog Rv0516c with its
anti-anti-anti-␴ factor, and of Rv2175c, a DNA-binding protein
of unknown function, with DNA, are abrogated upon phosphor-
ylation by kinases (39, 41), whereas transcription factor VirS
binds more strongly to its target promoter region (28). Func-
tions of cell division proteins Wag31 and FtsZ are also modu-
lated by phosphorylation (42, 43) The activity of enzymes like
GlmU, KasA, FabH, and MabA is down-regulated upon phos-
phorylation, although the action of KasB in the phosphorylated
form is enhanced (24, 26, 27, 29). Quemard et al. (11) in 1995
demonstrated activity of InhA by an indirect method in which
the action of InhA is measured by monitoring NADH oxidation
that occurs concurrently with substrate reduction. Upon per-
forming time course assays and kinetics experiments with InhA
and p-InhA, we observed a stark difference, with phosphoryla-
tion appearing to down-regulate the enzymatic efficiency of
InhA (Fig. 6).
Because the phosphorylation of InhA results in decreased
activity, we hypothesized that the overexpression of InhA and
kinase may impact the growth of mycobacteria, due to hyper-
phosphorylation of InhA. PknA overexpression was demon-
strated by to be detrimental to the growth (18). This is most
likely due to hyperphosphorylation of a number of substrates,
including InhA. We observed that overexpression of InhA and
PknA significantly compromised the growth over and above the
effects observed due to overexpression of PknA alone (Fig. 7).
Despite our best efforts, we were unable to co-express PknB and
InhA in M. smegmatis. It is possible that the deleterious effects
on mycobacterial growth upon co-expression of PknB and
InhA were so severe that cells overexpressing both proteins
may not have survived. Furthermore, in line with these results,
we found that the strain expressing InhA-T266E has a delayed
growth phenotype compared with the strain expressing the
wild type form of the protein (Fig. 7). Most importantly, InhA-
T266E mutant failed to functionally complement the InhA con-
ditional gene replacement mutant (13), strengthening our
hypothesis (Fig. 8). In addition, co-expression of InhA-T266A
mutant alongside PknA was found to partially alleviate the
diminution in growth (Fig. 7), emphasizing the importance
of phosphorylation at Thr-266. The fact that InhA-T266A mu-
tant ⫹ PknA overexpression did not bring growth back to
PknA-only overexpression levels suggests that, in addition to
Thr-266, PknA also targets other sites such as Thr-253 and
Thr-254 in vivo. InhA is shown to function as a dimer. Hence, it
is likely that hyperphosphorylated InhA (when the kinase is
overexpressed) is behaving like a dominant negative enzyme by
titrating away unphosphorylated InhA.
Based on our data, we conclude that InhA is a target of mul-
tiple mycobacterial STPKs, and the resulting phosphorylations
are instrumental in negatively modulating the growth of the
bacterium by virtue of decreased activity of this enzyme. In
depth studies by other groups have shown that other members
37870 JOURNAL OF BIOLOGICAL CHEMISTRY
of the FAS-II pathway, namely FabD, KasA, KasB, and MabA, as
well as FabH that links the FAS-I and FAS-II pathways, are also
regulated by phosphorylation attributed to these kinases (24,
26, 27). The identification of InhA as a novel substrate being
phosphorylated by multiple STPKs, along with other FAS
enzymes, is indicative of the various permutations and combi-
nations by which the mycolic acid synthesis is regulated in vivo.
A blend of multiple kinases, multiple substrates, and multiple
levels of fine-tuning of enzymatic activity based on phosphor-
ylation status puts forth an elegant and elaborate manner in
which mycolic acid synthesis can be tightly restrained, in tan-
dem with a multitude of intracellular and extracellular cues in
the bacteria.
Acknowledgments—We thank Dr. Tanya Parish and Dr. Jacobs, Jr.,
for providing pJAM2 and pMV-261apra vectors, respectively.
REFERENCES
1. Barry, C. E., 3rd, Lee, R. E., Mdluli, K., Sampson, A. E., Schroeder, B. G.,
Slayden, R. A., and Yuan, Y. (1998) Prog. Lipid Res. 37, 143–179
2. Takayama, K., Wang, C., and Besra, G. S. (2005) Clin. Microbiol. Rev. 18,
81–101
3. Bloch, K. (1977) Adv. Enzymol. Relat. Areas Mol. Biol. 45, 1– 84
4. Smith, S., Witkowski, A., and Joshi, A. K. (2003) Prog. Lipid Res. 42,
289 –317
5. Asselineau, J., and Lederer, E. (1950) Nature 166, 782–783
6. Kremer, L., Nampoothiri, K. M., Lesjean, S., Dover, L. G., Graham, S.,
Betts, J., Brennan, P. J., Minnikin, D. E., Locht, C., and Besra, G. S. (2001)
J. Biol. Chem. 276, 27967–27974
7. Choi, K. H., Kremer, L., Besra, G. S., and Rock, C. O. (2000) J. Biol. Chem.
275, 28201–28207
8. Sacco, E., Covarrubias, A. S., O’Hare, H. M., Carroll, P., Eynard, N., Jones,
T. A., Parish, T., Daffé, M., Bäckbro, K., and Quémard, A. (2007) Proc.
Natl. Acad. Sci. U.S.A. 104, 14628 –14633
9. Marrakchi, H., Lanéelle, G., and Quémard, A. (2000) Microbiology 146,
289 –296
10. Banerjee, A., Dubnau, E., Quemard, A., Balasubramanian, V., Um, K. S.,
Wilson, T., Collins, D., de Lisle, G., and Jacobs, W. R., Jr. (1994) Science
263, 227–230
11. Quémard, A., Sacchettini, J. C., Dessen, A., Vilcheze, C., Bittman, R., Ja-
cobs, W. R., Jr., and Blanchard, J. S. (1995) Biochemistry 34, 8235– 8241
12. Vilchèze, C., Morbidoni, H. R., Weisbrod, T. R., Iwamoto, H., Kuo, M.,
Sacchettini, J. C., and Jacobs, W. R., Jr. (2000) J. Bacteriol. 182, 4059 – 4067
13. Bhatt, A., Kremer, L., Dai, A. Z., Sacchettini, J. C., and Jacobs, W. R., Jr.
(2005) J. Bacteriol. 187, 7596 –7606
14. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D.,
Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., 3rd, Tekaia, F., Badcock,
K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Dev-
lin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels,
K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J.,
Quail, M. A., Rajandream, M. A., Rogers, J., Rutter, S., Seeger, K., Skelton,
J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S., and
Barrell, B. G. (1998) Nature 393, 537–544
15. Av-Gay, Y., and Everett, M. (2000) Trends Microbiol. 8, 238 –244
16. Chao, J., Wong, D., Zheng, X., Poirier, V., Bach, H., Hmama, Z., and Av-
Gay, Y. (2010) Biochim. Biophys. Acta 1804, 620 – 627
17. Alber, T. (2009) Curr. Opin. Struct. Biol. 19, 650 – 657
18. Kang, C. M., Abbott, D. W., Park, S. T., Dascher, C. C., Cantley, L. C., and
Husson, R. N. (2005) Genes Dev. 19, 1692–1704
19. Cowley, S., Ko, M., Pick, N., Chow, R., Downing, K. J., Gordhan, B. G.,
Betts, J. C., Mizrahi, V., Smith, D. A., Stokes, R. W., and Av-Gay, Y. (2004)
Mol. Microbiol. 52, 1691–1702
20. Walburger, A., Koul, A., Ferrari, G., Nguyen, L., Prescianotto-Baschong,
C., Huygen, K., Klebl, B., Thompson, C., Bacher, G., and Pieters, J. (2004)
VOLUME 285 • NUMBER 48 • NOVEMBER 26, 2010
 Regulation of Mycobacterial Enoyl-ACP Reductase InhA
Science 304, 1800 –1804
21. Tiwari, D., Singh, R. K., Goswami, K., Verma, S. K., Prakash, B., and Nandi-
coori, V. K. (2009) J. Biol. Chem. 284, 27467–27479
22. Papavinasasundaram, K. G., Chan, B., Chung, J. H., Colston, M. J., Davis,
E. O., and Av-Gay, Y. (2005) J. Bacteriol. 187, 5751–5760
23. Deol, P., Vohra, R., Saini, A. K., Singh, A., Chandra, H., Chopra, P., Das,
T. K., Tyagi, A. K., and Singh, Y. (2005) J. Bacteriol. 187, 3415–3420
24. Molle, V., Brown, A. K., Besra, G. S., Cozzone, A. J., and Kremer, L. (2006)
J. Biol. Chem. 281, 30094 –30103
25. Bhatt, A., Molle, V., Besra, G. S., Jacobs, W. R., Jr., and Kremer, L. (2007)
Mol. Microbiol. 64, 1442–1454
26. Veyron-Churlet, R., Zanella-Cléon, I., Cohen-Gonsaud, M., Molle, V., and
Kremer, L. (2010) J. Biol. Chem. 285, 12714 –12725
27. Veyron-Churlet, R., Molle, V., Taylor, R. C., Brown, A. K., Besra, G. S.,
Zanella-Cléon, I., Fütterer, K., and Kremer, L. (2009) J. Biol. Chem. 284,
6414 – 6424
28. Kumar, P., Kumar, D., Parikh, A., Rananaware, D., Gupta, M., Singh, Y.,
and Nandicoori, V. K. (2009) J. Biol. Chem. 284, 11090 –11099
29. Parikh, A., Verma, S. K., Khan, S., Prakash, B., and Nandicoori, V. K. (2009)
J. Mol. Biol. 386, 451– 464
30. Triccas, J. A., Parish, T., Britton, W. J., and Gicquel, B. (1998) FEMS Mi-
crobiol. Lett. 167, 151–156
31. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol.
201, 110 –149
NOVEMBER 26, 2010 • VOLUME 285 • NUMBER 48
32. Parish, T., and Stoker, N. G. (1998) Methods Mol. Biol. 101, 129 –144
33. Eblen, S. T., Kumar, N. V., Shah, K., Henderson, M. J., Watts, C. K., Shokat,
K. M., and Weber, M. J. (2003) J. Biol. Chem. 278, 14926 –14935
34. He, X., Alian, A., Stroud, R., and Ortiz de Montellano, P. R. (2006) J. Med.
Chem. 49, 6308 – 6323
35. DeLano, W. L. (2002) The PyMOL Molecular Graphies System, DeLano,
Scienctific LLC, San Carlos, CA
36. Rozwarski, D. A., Vilchèze, C., Sugantino, M., Bittman, R., and Sacchettini,
J. C. (1999) J. Biol. Chem. 274, 15582–15589
37. Molle, V., and Kremer, L. (2010) Mol. Microbiol. 75, 1064 –1077
38. Canova, M. J., Kremer, L., and Molle, V. (2009) J. Bacteriol. 191,
2876 –2883
39. Greenstein, A. E., MacGurn, J. A., Baer, C. E., Falick, A. M., Cox, J. S., and
Alber, T. (2007) PLoS Pathog. 3, e49
40. Sharma, K., Gupta, M., Krupa, A., Srinivasan, N., and Singh, Y. (2006)
FEBS J. 273, 2711–2721
41. Cohen-Gonsaud, M., Barthe, P., Canova, M. J., Stagier-Simon, C., Kremer,
L., Roumestand, C., and Molle, V. (2009) J. Biol. Chem. 284, 19290 –19300
42. Kang, C. M., Nyayapathy, S., Lee, J. Y., Suh, J. W., and Husson, R. N. (2008)
Microbiology 154, 725–735
43. Thakur, M., and Chakraborti, P. K. (2006) J. Biol. Chem. 281,
40107– 40113
44. Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T., and Jacobs, W. R., Jr.
(1990) Mol. Microbiol. 4, 1911–1919
JOURNAL OF BIOLOGICAL CHEMISTRY 37871