ArticlePDF Available

Post-Translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis

Authors:

Abstract and Figures

Iron is an essential element for life, but its soluble form is scarce in the environment and is rarer in the human body. Mycobacterium tuberculosis (Mtb) produces two aryl-capped siderophores, mycobactin (MBT) and carboxymycobactin (cMBT), to chelate intracellular iron. The adenylating enzyme MbtA catalyzes the first step of mycobactin biosynthesis in two half-reactions: activation of the salicylic acid as an acyl-adenylate and ligation onto the acyl carrier protein (ACP) domain of MbtB to form covalently salicylated MbtB-ACP. We report the first apo-MbtA structure from Mycobacterium smegmatis at 2.3 Å. We demonstrate here that MbtA activity can be reversibly, post-translationally regulated by acetylation. Indeed the mycobacterial protein acetyltransferase (Pat), Rv0998, specifically acetylates MbtA on lysine 546, in a cAMP-dependent manner, leading to enzyme inhibition. MbtA acetylation can be reversed by the NAD+-dependant deacetyltransferase, Rv1151c (DAc). Deletion of Pat and DAc genes in Mtb revealed distinct phenotypes for strains lacking one or the other genes at low pH and limiting iron conditions. This study establishes a direct connection between the reversible acetylation system Pat/DAc and the ability of Mtb to adapt in limited iron conditions, which is critical for mycobacterial infection.
Content may be subject to copyright.
Mycobactin MbtA regulation!
!
1!
Post-Translational Acetylation of MbtA Modulates Mycobacterial Siderophore Biosynthesis*.
Olivia Vergnolle,1 Hua Xu,1 JoAnn M. Tufariello,2 Lorenza Favrot,1 Adel A. Malek,2 William R.
Jacobs Jr2 and John S. Blanchard1
1Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
New York 10461, USA
2Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein
College of Medicine, Bronx, New York 10461, USA
*Running title: Mycobactin MbtA regulation
To whom correspondence should be addressed: John S. Blanchard, Department of Biochemistry, Albert
Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA, Tel.: +001 718
430 3096; E-mail: john.blanchard@einstein.yu.edu
Keyword: MbtA, acetylation, structure, mycobactin, Mycobacterium tuberculosis
Iron is an essential element for life, but its
soluble form is scarce in the environment and is
rarer in the human body. Mycobacterium
tuberculosis (Mtb) produces two aryl-capped
siderophores, mycobactin (MBT) and
carboxymycobactin (cMBT), to chelate
intracellular iron. The adenylating enzyme
MbtA catalyzes the first step of mycobactin
biosynthesis in two half-reactions: activation of
the salicylic acid as an acyl-adenylate and
ligation onto the acyl carrier protein (ACP)
domain of MbtB to form covalently salicylated
MbtB-ACP. We report the first apo-MbtA
structure from Mycobacterium smegmatis at 2.3
Å. We demonstrate here that MbtA activity can
be reversibly, post-translationally regulated by
acetylation. Indeed the mycobacterial protein
acetyltransferase (Pat), Rv0998, specifically
acetylates MbtA on lysine 546, in a cAMP-
dependent manner, leading to enzyme
inhibition. MbtA acetylation can be reversed by
the NAD+-dependant deacetyltransferase,
Rv1151c (DAc). Deletion of Pat and DAc genes
in Mtb revealed distinct phenotypes for strains
lacking one or the other genes at low pH and
limiting iron conditions. This study establishes
a direct connection between the reversible
acetylation system Pat/DAc and the ability of
Mtb to adapt in limited iron conditions, which is
critical for mycobacterial infection.
Iron is the most abundant element in the
earth’s core and represent 5% of the earth crust
(1). However, because ferric iron, Fe(III), is
poorly soluble in water at neutral pH, its biological
availability is scarce (10-18 M in water) (2). Iron is
a vital nutrient for most prokaryotes and
eukaryotes with a notable exception among well
studied pathogenic organisms being the Lyme
disease pathogen, Borrelia burgdorferi, which
uses manganese (3). Iron is required for DNA
replication, oxygen transport, energy generation,
enzymatic redox reactions and oxidative stress
protection. Due to the scarcity of free iron,
essentially all living species have evolved ways to
acquire and store iron and regulate iron levels. In
humans, the majority of iron is found in the
porphyrin ring of heme proteins, in iron/sulfur-
containing enzymes or sequestered in the ferritin
protein. To prevent pathogen colonization, humans
deplete free iron availability to a concentration of
10-24 M in serum using the transferrin protein (4)
and macrophages decrease the expression of
ferritin and the transferrin receptor to keep
intracellular macrophage iron levels low (5). To
circumvent host protection mechanisms, the
intracellular pathogen Mycobacterium tuberculosis
(Mtb) has fine-tuned an effective iron-chelating
arsenal during its 70,000 year old cohabitation
with humans (6). Mtb acquires iron in two distinct
ways: a high-affinity heme acquisition system
(7,8) and via a siderophore-based system (9-11).
Mtb synthesizes two related aryl-capped
polyketide-polypeptide siderophores, called
mycobactin (MBT) and carboxymycobactin
(cMBT) (Fig. 1A) (12). These siderophores differ
only in the composition of their aliphatic tails,
http://www.jbc.org/cgi/doi/10.1074/jbc.M116.744532The latest version is at
JBC Papers in Press. Published on August 26, 2016 as Manuscript M116.744532
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
2!
which determine the localization of the
siderophore: long-chain fatty acylated MBT is
membrane-associated while cMBT contains short-
chain dicarboxy-fatty acids and is more water-
soluble and is secreted by the pathogen (13,14).
Two MBT gene clusters, Mbt-1 and Mbt-2 (Fig.
1B), are composed of a mixture of nonribosomal
peptide synthetase and polyketide synthase
enzymes (15). Three specific enzymes, MbtA,
MbtM (also known as FadD33) and MbtK are
essential for mature mycobactin biosynthesis (16).
MbtA catalyzes the first step of MBT core
synthesis by activating salicylic acid as an
acyladenylate before subsequent ligation to the
pantothenyl group of the acyl carrier protein
domain of MbtB (Fig. 1C) (12). FadD33 activates
the long chain fatty acid before its transfer onto
the central N-hydroxy lysine residue of the MBT
core (16). MbtK is a GNAT N-acetyltransferase
that transfers the long chain fatty acid onto the
mycobactin core (16).
Iron-dependent transcriptional regulators,
IdeR and HupB, control the transcription level of
genes involved in iron metabolism including Mbt-
1 and Mbt-2 in order to avoid excessive iron
uptake, storage and toxic overload in the bacterial
cell (17,18). Another level of MBT regulation was
recently characterized as a post-translational
acetylation of FadD33 by the protein lysine
acetyltransferase (Pat) in Mycobacterium
smegmatis (Msmeg) (19). Pat acetylates and
inactivates FadD33 in a cAMP-dependent manner,
which is then reversed by a NAD+-dependent
Sirtuin-like deacetylase, DAc1, to restore FadD33
activity. Pat has been structurally characterized
and consists of an N-terminal cAMP-binding
domain fused to a C-terminal GCN5-related N-
acetyl-transferase domain (GNAT) (20,21). The
N-terminal domain allows Pat to be activated by
the second messenger, cAMP, produced by the
adenylate cyclase (AC) enzymes. The Mtb genome
encodes a total of 16 ACs, which are sensitive to
diverse environmental stimuli, including pH,
hypoxia, fatty acids and carbon dioxide level (22).
This complex response system allows
mycobacteria to rapidly adapt to external changes
by relaying this information, via a specific cAMP
pathway, to downstream effectors.
In this study, we demonstrate that the first
committed step in mycobactin biosynthesis,
catalyzed by MbtA, can also be reversibly post-
translationally acetylated by Pat with the loss of its
enzymatic activity, and reactivated by DAc
through deacetylation. Deletion of Pat and DAc
genes in Mtb highlights the importance of those
specific two genes for normal MBT production
during iron starvation.
RESULTS
Cloning, Expression, and Purification of MbtA. To
avoid solubility problems, Mtb and Msmeg N-
terminal-His6-tagged MbtA were expressed at low
temperature. Heterologous expression in E. coli
gave good quantities of soluble MbtA. After
purification by nickel affinity chromatography an
apparent molecular weight of ~59 kDa was
observed by SDS-PAGE, which is consistent with
the predicted 58983 Da and 59280 Da molecular
weight for Msmeg and Mtb respectively. Mtb and
Msmeg MbtA share 69.2 % sequence identity and
85.0 % sequence similarity.
Cloning, Expression, and Purification of MbtB.
The Mtb ACP domain of MbtB module was
expressed in E. coli but was mostly insoluble.
Using denaturing conditions, small quantities of
soluble apo-MbtB-ACP were obtained after nickel
affinity chromatography. As assessed by SDS-
PAGE, apo-MbtB-ACP displayed an apparent
molecular mass of 11 kDa, consistent with the
molecular weight of 11670 Da calculated from the
amino acid sequence. For the subsequent activity
assay with MbtA, apo-MbtB-ACP is required to be
phosphopantetheinylated (holo-MbtB-ACP form).
Sfp, a phosphopantetheinyl transferase from
Bacillus subtilis (B. subtilis) was used to
covalently transfer the 4’-phosphopantetheinyl
group from coenzyme A onto apo-MbtB-ACP as
described in the Materials and Methods section.
The conversion of apo-MbtB-ACP to holo-MbtB-
ACP was confirmed by addition of 340 Da moiety,
corresponding to the phosphopantetheine arm by
Fourier transform mass spectral analysis (data not
shown).
Specific acetylation of MbtA by protein lysine
acetyltransferase. Recently, protein lysine
acetyltransferase (Pat) has been identified as a
regulator of one of the later step of mycobactin
biosynthesis via post-translational modification of
fatty acyl-AMP ligase FadD33 (19). MbtA and
FadD33 both catalyze an adenylation reaction
followed by thioesterification of the substrate onto
an ACP. FadD33 is acetylated by Pat on K511 and
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
3!
amino acid sequence comparison of MbtA and
FadD33 highlights a conserved lysine residue,
K546, in MbtA (Fig. 2A). Moreover like FadD33-
K511, MbtA-K546 is also flanked by a preceding
glycine as well as two nearby downstream basic
residues, which are believed to lower the steric
hindrance between Pat and MbtA and reduce the
pKa value of the NH2 group of K546 respectively
(Fig. 2A). Based on these observations we probed
MbtA as a potential substrate for Pat. For this
experiment Msmeg Pat (MSMEG_5458) was used
instead of Mtb Pat (Rv0998) since protein
refolding issues occurred after protein purification
therefore affecting Mtb Pat activity. Western-blot
analysis using anti-acetyllysine antibody shows
that MbtA is acetylated by Pat in the presence of
acetyl-CoA and cAMP (Fig. 2B, lane 2). Negative
controls without Pat or acetyl-CoA show no MbtA
acetylation (Fig. 2B, lanes 3 and 4). Only in the
negative control without cAMP, a light MbtA
acetylation is observed (Fig. 2B, lane 5) likely due
to co-purification of Pat with some bound cAMP
as noted previously (23). Msmeg Pat also
acetylates Mtb MbtA in the same manner (data not
shown). To determine the site(s) of acetylation we
created a single amino acid change in MbtA,
changing K546 to an alanine. K546 appears to be
the main acetylation site since the K546A mutant
completely loses the ability to be acetylated by Pat
by western-blot (Fig.2B, lane 1). To confirm this
unique acetylation site, in vitro acetylated MbtA
was analyzed by mass spectrometry. Good
sequence coverage (85%) allowed the
identification of K546 as the single acetylation site
(Fig. 2C) and confirms that Pat acetylates MbtA
on K546.
Effect of Acetylation on MbtA Activity. MbtA
enzymatic activity was followed by monitoring the
formation of AMP in the presence of 2,3-
dihydroxybenzoate and acceptor holo-MbtB-ACP.
To test acetylation effects, the activity of Msmeg
MbtA was monitored overtime in the presence of
Msmeg Pat, acetyl-CoA and cAMP. After 5 hrs of
incubation, Msmeg MbtA activity was reduced by
80 % whereas MbtA activity without either acetyl-
CoA or Pat remained unchanged (Fig. 3A). The
gradual loss of activity is directly mediated by
MbtA acetylation in a Pat- and acetylCoA-
dependent manner. Using the same Mtb MbtA and
Msmeg Pat ratio as used previously for Msmeg
MbtA, 12 hrs of incubation were necessary to
achieve complete Mtb MbtA inactivation (data not
shown). For all subsequent experiments MbtAs
were incubated overnight with Msmeg Pat to
insure full inactivation. The Mtb genome encodes
one known sirtuin-like deacetylase DAc
(Rv1151c) in contrast to the Msmeg genome,
which contains two sirtuin-like deacetylases
named DAc1 (Msmeg_5175) and DAc2
(Msmeg_4620). DAc and DAc1 are very similar
and were previously shown to deacetylate
acetylated forms of acetyl-CoA synthetase (ACS)
and FadD33 (19,23). Western-blot analysis of
acetylated Mtb and Msmeg MbtA after overnight
incubation with DAc deacetylase demonstrates
that both MbtAs are deacetylated by DAc (Fig.
4A, lane 2 and 4B, lane 3). To check if
deacetylation restores MbtA activity, acetylated
MbtA was incubated with DAc and NAD+. Over
time, inactive acetylated MbtA regained its
activity when incubated with NAD+ and DAc.
However, omission of either NAD+ or deacetylase
prevented reactivation (Fig. 3B). These results
suggest that MbtA catalytic activity is regulated by
Pat and DAc via the reversible acetylation and
deacetylation of MbtA at position K546.
Crystal structure of Msmeg MbtA apo. Msmeg
MbtA was crystallized in an apo form and the
structure was solved to 2.3 Å (Table 1). The
Msmeg MbtA structure was solved by molecular
replacement using the DhbE structure (accession
code: 1MDB), which is the adenylation domain in
the bacillibactin siderophore biosynthesis (24).
Molecular replacement led to a partial solution for
MbtA with a defined N-terminal domain and two
C-terminal domain fragments (residues 450-462
and 478-503). To complete the Msmeg MbtA
structure, the C-terminal domain of DhbE was
aligned with the MbtA C-terminal fragments.
Through multiple cycles of refinements and
manual corrections, a Msmeg MbtA model was
generated containing one chain in the asymmetric
unit with residues 18-151, 157-313, 321-556
observed and with a Rwork of 18.9 % and a Rfree of
24.5 %. The overall structure of Msmeg MbtA
exhibits the typical fold observed for the ANL
(Acyl/Aryl, NonRibosomal Peptide Synthetases
and Luciferase) superfamily of adenylating
enzymes (25). The structure includes two main
domains (Fig. 5A): a large N-terminal domain (1-
457) and a smaller C-terminal domain (458-558).
The N-terminal domain is composed of three
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
4!
subdomains: two β-sheet subdomains (a and b)
and a β-barrel subdomain (c). The subdomains (a)
and (b) form a five-layered αβαβα sandwich. The
subdomain (a) includes six β-strands and five α-
helices while the second β-sheet subdomain
contains eight β-strands and seven α-helices. The
β-barrel subdomain (c) abuts (a) and (b) and leads
to the compact C-terminal domain through a short
hinge (including residue K457). The compact C-
terminal domain consists of three α-helices and
five β-strands and harbors K546, the lysine
acetylated by Pat.
The Msmeg MbtA structure is very similar to the
B. subtilis DhbE (44 % sequence identity) and the
Acinetobacter baumannii BasE N-terminal domain
(39 % sequence identity) structures.
Superimposition of MbtA with DhbE and BasE N-
terminal domain shows low r.m.s displacement
values for the Cα atom positions of 1.0 Å and 0.7
Å respectively (Fig. 5B) (24,26). The highly
conserved P-loop motif (S212-K221) is implicated
in the ATP phosphate moiety binding and adopts a
slightly different conformation compared to the
BasE N-terminal domain or the DhbE structures
due to the lack of substrate in the MbtA active site
(24,26). Salmonella enterica acetyl-CoA
synthetase (ACS) and MbtA both catalyzed
adenylation reactions and structural
superimposition indicates a fairly high structure
similarity (4.9 Å r.m.s displacement for the Cα
atom positions) (Fig. 5C) (27). Moreover, Pat
acetylates ACS-K609 and MbtA-K546, which are
both localized on the same C-terminal domain
loop. The main difference between the Msmeg
MbtA structure and the other structures (DhbE and
ACS) lies in the conformation adopted by the C-
terminal domain: the latter is rotated by 91° and
102° compared to the DhbE and the ACS C-
terminal domains, respectively. The C-terminal
domain forms a “lid”, which can close above the
β-barrel subdomain and plays a significant role in
the transfer of the adenylated product to the acyl-
carrier protein (24,28). This domain has been
shown to adopt different orientations depending on
which half-reaction is catalyzed, either the
adenylation or the thioesterification reactions
(25,29).
Generation of precise null deletion mutants in
genes encoding Pat (Rv0098) and DAc (Rv1151c)
in Mtb. Prior to this study, the potential for
regulation of mycobacterial siderophore
production via post-translational acetylation of
enzymes required for mycobactin biosynthesis has
been demonstrated biochemically. Here, we also
examine the in vivo impact of genetically
removing Pat or Dac (Fig. 6A and 6C), under
conditions encountered in the intraphagosomal
environment. Towards this end, using a BLS2-safe
M. tuberculosis H
37Rv derivative, which is a
double auxotroph for leucine and pantothenate, we
engineered Pat and DAc Mtb mutant strains
(Fig. 6B and 6D). The genes were deleted by the
specialized transduction methodology described in
the Materials and Methods. To verify the
deletions, PCR amplification using three
diagnostic independent primer pairs shows
expected amplicon sizes for Pat + pMV261 and
DAc + pMV261 (Fig. 6E and 6F).
Pat and DAc share distinct phenotypes at low
pH and limited iron conditions. Pat exhibits a
unique structural feature with an N-terminal
cAMP-binding domain fused to a C-terminal
GNAT domain. Our current study and past studies
(19,23) have shown that Pat acetylation activity is
dependent on cAMP generated by multiple
adenylate cyclases (AC). To investigate the effect
of Pat and DAc deletions in Pat and DAc, we
tested these strains under different physiological
conditions such as low pH at which specific ACs
induce cAMP production (22). As a reference
point, we first determined growth curves for the
five strains in Sauton medium at pH 7. We
observed no significant growth difference between
the Mtb WT + pMV261 (expression vector), Mtb
Pat + pMV261, Mtb DAc + pMV261, Mtb
Pat + pMV261-Pat and Mtb DAc + pMV261-
DAc (Fig. 7A). All strains carried the pMV261
expression vector in order to facilitate a
comparative analysis. In contrast, when these same
strains were grown in Sauton medium at pH 6,
Mtb Pat + pMV261 showed a growth advantage
compared to the parent and complemented strains,
while Mtb DAc + pMV261 displayed a slight
growth defect (Fig. 7B). These results indicate that
deletion of Pat leads to an acceleration of growth
and suggests that lack of acetylation of Pat
substrates at pH 6 is beneficial for Mtb. Since Pat
is able to potentially inhibit mycobactin
biosynthetic enzymes in vivo, we next cultured the
five strains in Sauton medium with limiting iron
(100 fold less than standard Sauton) to test the
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
5!
same hypothesis that inhibition of siderophore
production under these conditions would inhibit
growth in vivo. In iron-limited Sauton medium at
pH 7, we observed no significant difference in the
growth of the strains, but a much slower growth
rate was noted as compared with iron-sufficient
media (Fig 7C). If we now compared the growth
pattern in Sauton pH 6 with limited iron, we
observed a distinct growth advantage for Mtb Pat
+ pMV261 compared to the parent and
complemented stains, while Mtb DAc + pMV261
displayed a major growth defect (Fig. 7D). These
results support the idea that in vivo, at lower pH
and under elevated cAMP levels, that deletion of
Mtb Pat likely prevents the acetylation, and
inhibition of MbtA and FadD33, thus allowing for
mycobactin production, while deletion of Mtb
DAc likely prevents the deacetylation of these
enzymes and inhibits mycobactin production.
DISCUSSION
The acquisition of iron from the
environment, its incorporation into iron-containing
proteins and enzymes and the regulation of
intracellular iron levels is essential for the growth
and virulence of Mtb. While several methods for
iron acquisition exist in the organism, the major
mechanism is the production of the iron
siderophore, mycobactin. This secondary
metabolite has a number of unusual features. A
hydroxyphenyloxazoline moiety, made from
salicylate and serine, forms a N-cap to the adjacent
εN-hydroxylysine residue that is additionally
acylated on the ε-amino group. The remainder of
the molecule contains a hydroxybutyryl group and
a terminal εN-hydroxylysine residue that is
cyclized into a seven-membered hydroxamate
ring. The hexadentate coordination of ferric iron
occurs from the six oxygen and nitrogen atoms in
the molecule shown in blue in Figure 1A.
Acylation of the central hydroxylysine residue
occurs with saturated and Δ2-unsaturated long
chain fatty acids to generate mycobactin or with
short chain dicarboxylic acids to generate
carboxymycobactins. It is thought that the
carboxymycobactins are secreted and acquire host
iron and then deliver this iron to the membrane-
associated mycobactins (10).
It has been recognized for some time that
bacteria tightly regulate the production of
siderophores such as mycobactin. In Mtb, a major
regulator of mycobactin production is the iron-
binding transcriptional regulator IdeR (Rv2711).
This 230-residue protein is essential for
mycobacterial growth and is a member of the
diphtheria toxin repressor family and exists as a
homodimer in solution (30). It binds to a highly
conserved 19 base pair inverted repeat that is
found adjacent to genes whose transcription is
repressed at high iron concentrations. In both the
Mbt-1 and Mbt-2 gene clusters, multiple IdeR
binding sites are observed adjacent to the
transcriptional start sites for the genes in the two
clusters. As iron is acquired by the mycobactins
and the intracellular concentration increases, the
iron-bound IdeR binds to these regions, effectively
preventing transcription of these genes (31). This
is a general mechanism of regulation in many
bacteria. There is a recent report that the HupB
protein can serve as a positive regulator of
mycobactin synthesis under conditions of low
intracellular iron by binding to a region 5’ to that
of the IdeR binding site and recruiting RNA
polymerase to the transcription start site (32).
In the current study, we have analyzed the
potential for non-transcriptional regulation of
mycobactin biosynthesis. We have previously
reported the reversible, post-translational
monoacetylation of both the Mtb acetyl-CoA
synthetase (ACS) and the mbtM-encoded acyl-
ACP synthetase (FadD33) (19,23). These enzymes
are mechanistically related and use ATP to form
an intermediate acetyl or fatty acyl adenylate,
which is then nucleophilically attacked by the thiol
of CoASH or the phosphopantetheine arm of an
ACP domain. Knowing the primary sequence
around the lysine residue that is acetylated in both
these enzymes, we identified a highly similar
sequence in the Mtb and Msmeg MbtA enzymes. It
was previously shown that MbtA-K546 lysine
equivalents in FadD33 and ACS are required for
catalysis in the first adenylation half reaction
(19,23). The mechanism of MbtA is similar to that
of both ACS and FadD33 in that a carboxyl group
of the substrate is initially adenylated for
activation, followed by the attack of a thiol group
on the mixed carboxylic-phosphoric anhydride,
although MbtA uniquely uses an aryl carboxylate,
salicylate. We now show that MbtA is also
reversibly acetylated and that this leads to enzyme
activity loss.
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
6!
MbtA has become a well-established
druggable target in the last decade. The production
of mycobactin is essential and the tight binding of
hydrolytically stable, isosteric analogs of the
salicyladenylate has been demonstrated (33). The
intermediate analog, 5’-O[N-
(salicyl)sulfamoyl]adenosine was shown
independently by three groups to inhibit MbtA
with a Ki of ~ 6 nM (33-35). This compound
exhibits antimycobacterial activity in vitro with a
MIC value of 0.4 mM, and exhibited in vivo
activity in an acute mouse model. Since MbtA is
the first and committed step in mycobactin
synthesis, it is perhaps not surprising that its
inhibition would be so deleterious.
To support our biochemical analysis we
also performed genetic analysis to investigate the
role of acetylation in the Mtb physiological
contest. We generated two strains in which either
the Pat or DAc genes were knocked-out, and
strains in which the knockouts were
complemented. To mimic the environment inside
the macrophage phagosome, strains were grown at
low pH and limiting iron condition. While at pH 7
(Sauton pH7 and limited iron pH7), all strains
exhibit a similar growth phenotype. But at relevant
physiological pH (pH 6 and under limiting iron
conditions), the ΔPat strain grows significantly
faster than the WT, and the ΔDAc grows
considerably slower. Both complemented strains
grow approximately as well as WT. This suggests
that when acetylation of MbtA or FadD33 is
prevented in the ΔPat strain, the resulting active
enzymes are capable to generating the required
levels of mycobactin. On the other hand, in the
ΔDAc strain, MbtA and FadD33 are likely to be
functioning at less than full activity, even less
activity than the WT strain resulting in insufficient
mycobactin levels. Thus mutant phenotypes
suggest that acetylation may influence Mtb
survival in the macrophage.
It is clear that the transcriptional
regulation of the mycobactin biosynthetic gene
clusters is the major mechanism by which
mycobactin biosynthesis is regulated. However,
once the proteins are made, they remain
constitutively active, potentially enabling for the
production of excess mycobactin, and allowing for
the accumulation of intracellular iron in excess of
its ability to be incorporated into iron-dependent
proteins. This would lead to free iron in the cell,
and in the reducing, but aerobic, intracellular
environment of the cell, the potential for the
generation of reactive oxygen species through the
action of Fenton-type chemistry. In support of this,
a recent report showed that Mtb is particularly
sensitive to ascorbic acid, Vitamin C, in
comparison to other Gram negative and positive
species (36). The ability to rapidly inactivate
MbtA could be used to break the ATP-consuming
production of mycobactin very quickly when the
organism senses that sufficient iron is present.
This secondary level of regulation could be used to
fine tune iron levels, allowing just enough as
required and preventing the over accumulation of
iron to toxic levels.
EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of MbtA.
MbtA (MSMEG_4516 and Rv2384) was
amplified from Msmeg mc
2155 and Mtb H37Rv
genomic DNA using the primer pairs
Msmeg_MbtA_F (5’-
GGAATTCCATATGACTCTGACCACGCCCC
AC-3’) and Msmeg_MbtA_R (5’-
CCCAAGCTTCCCGCCGAGCTGACGCACGA-
3’) or Mtb_MbtA_F (5’-
GGAATTCCATATGCCACCGAAGGCGGCAG
AT-3’) and Mtb_MbtA_R (5’-
CCCAAGCTTATGGCAGCGCTGGGTCGTCA
C-3’) containing NdeI and HindIII sites,
respectively. The PCR amplicon was ligated into
the pET-28a (+) vector, and then transformed into
E. coli DH5α competent cells to create pET-28a
(+)::MbtA N-terminally His6 tag plasmid. A
sequence verified construct was transformed in E.
coli T7 express lysy/Iq for protein expression. A 4
mL preculture was used to inoculate 1 L of Luria-
Bertani medium supplemented with 50 µg/mL
kanamycin. The culture was grown to midlog
phase (A600 ~ 0.6) at 37 °C, then induced by the
addition of 0.4 mM isopropyl-1-thio-β-D-galacto-
pyranoside (IPTG). After 18 hrs of additional
incubation (20 °C), cells were harvested by
centrifugation (6,000 g, 20 min) and stored at -80
°C. The cell pellet was thawed and resuspended in
lysis buffer (25 mM Tris, pH 8.0 containing 150
mM NaCl, 10 mM imidazole) supplemented with
DNaseI (0.1 µg/mL), lysozyme (2 mg/ml) and
EDTA-free protease inhibitors cocktail (Roche).
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
7!
Resuspended cells were disrupted by sonication
and cellular debris was removed from the lysate by
centrifugation at 16000 rpm for 50 min. MbtA was
purified by nickel affinity chromatography. After
an extensive wash with 25 mM Tris, pH 8.0
containing 150 mM NaCl and 10 mM imidazole
buffer, the bound protein was eluted with a linear
imidazole gradient (30-250 mM). Pure fractions,
as determined by SDS-PAGE, were pooled
together for buffer-exchange into 25 mM Tris, pH
8.0 containing 150 mM sodium chloride and 10 %
glycerol. For crystallization purpose, the N-
terminal polyhistidine tag of Msmeg MbtA was
cleaved using thrombin protease. The cleavage
reaction was dialyzed overnight against 50 mM
Tris (pH 8.0), 150 mM sodium chloride, 1 mM
dithiothreitol and 1 mM calcium chloride. The
enzyme was further purified using size exclusion
chromatography (HiLoad
TM 26/60 SuperdexTM 75
prep grade) in the buffer described above without
calcium chloride and then concentrated to 12
mg/mL using a 30-kDa Amicon Ultra Centrifugal
filter.
Crystallization. The apo enzyme was screened
against the MCSG suite (Microlytic, 384
conditions). A Crystal Gryphon robot (Art
Robbins) was used to dispense both the reservoir
solutions and the sitting-drops (1:1 ratio). Crystal
trays (Intelli-Plates 96, Art Robbins) were then
sealed and incubated at 20 °C. Preliminary crystals
were obtained in the condition 1D2 containing 0.2
M sodium chloride, 0.1 M bis-tris (pH 6.5) and 25
% (w/v) polyethylene glycol 3350. The
crystallization condition containing 0.1 M bis-tris
(pH 6.5), 0.4 M sodium chloride and 17.5 % (w/v)
polyethylene glycol 3350 improved the quality of
the crystal. The latter was cryoprotected by
addition of 30 % (w/v) polyethylene glycol and
flash-cooled in liquid nitrogen.
Data collection and structure determination.
Diffraction data were collected at the Lilly
Research Laboratories Collaborative Access Team
(LRL-CAT) beamline at the Advance Photon
Source Argonne National Laboratory (APS-ANL,
IL), at 0.97931 Å wavelength radiation.
Diffraction was observed to 2.3 Å. Data was
indexed, integrated and finally scaled using the
XDS package (37). Matthews’ coefficient analysis
indicated the presence of one molecule in the
asymmetric unit (43 % solvent). The structure of
DhbE from B. subtilis (PDB entry 1MDF) was
used to carry out molecular replacement with
PHASER-MR (24,38). The solution obtained by
molecular replacement was used as a template for
the Autobuild tool to build 78 % of the model (39).
The rest of the model was manually built using
COOT and iterative cycles of refinements (rigid
body refinement, simulated annealing, positional
and B-factor refinements) (40,41). Three residues
were observed in the disallowed region of the
Ramachandran plot (V197, V544, R554).
Cloning, Expression, and Purification of MbtB-
ACP. The acyl carrier protein domain of MbtB
enzyme, MbtB-ACP (Rv2383c) was amplified
from Mtb genomic DNA using the primer pairs
Mtb_MbtB-ACP_F (5’-
GGAGGGCATATGGTGCATGCTACGGCG-3’)
and Mtb_MbtB-ACP_R (5’-
CCCAAGCTTTGCGGCAACTGCCGTGGG-3’)
containing NdeI and HindIII sites, respectively.
The PCR amplicon was ligated into the pET-28a
(+) vector, and then transformed into E. coli
DH5α competent cells to create pET-28a
(+)::MbtB-ACP N-terminally His6 tag plasmid. A
sequence verified construct was transformed in E.
coli T7 express lys
y/Iq for protein expression.
MbtB-ACP domain was expressed as per MbtA
and cells were stored at -80 °C. The cell pellet was
thawed and resuspended in lysis buffer (25 mM
Tris, pH 8.0) supplemented with DNaseI (0.1
µg/mL) and EDTA-free protease inhibitors
cocktail (Roche). Resuspended cells were
disrupted by homogenization using the
EmulsiFlex-C3 (Avestin) at 10000 psi and cellular
debris was removed from the lysate by
centrifugation at 16000 rpm for 50 min. MbtB-
ACP was purified by nickel affinity
chromatography. After extensive wash with 4 M
urea, 25 mM Tris, pH 8.0, 10 mM imidazole
buffer, the bound protein was eluted with a linear
imidazole gradient (30-250 mM) with 4M urea.
Pure fractions, as determined by SDS-PAGE were
pooled together for buffer-exchange first in 1 M
urea, 50 mM Tris, pH 8.0, 150 mM NaCl and then
in 50 mM Tris, pH 8.0, 150 mM NaCl to allow
refolding. MbtB-ACP was concentrated using an
Amicon pressure concentrator with a 3 K Dalton
filter to 100 µM.
Cloning, Expression, and Purification of Sfp. Sfp
was amplified from B. subtilis genomic DNA
using the primer pairs Bsub_Sfp_F (5’-
GGTATTGAGGGTCGCATGAAGATTTACGG
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
8!
AATT-3’) and Bsub_Sfp_R (5’-
AGAGGAGAGTTAGAGCCTTATAAAAGCGC
TTCGTA-3’). The PCR amplicon was ligated into
the pET-30 Xa/LIC (+) vector, and then
transformed into E. coli DH5α competent cells to
create pET-30 Xa (+)::Sfp N-terminally His6 tag
plasmid. A sequence verified construct was
transformed in E. coli T7 express lysy/Iq for protein
expression. A 4 mL preculture was used to
inoculate 1 L of Luria-Bertani medium
supplemented with 50 µg/mL kanamycin. The
culture was grown to midlog phase (A600 ~ 0.6) at
37 °C, then induced by the addition of 0.1 mM
IPTG. After 18 hrs of additional incubation (30
°C), cells were harvested by centrifugation (6,000
g, 20 min) and stored at -80 °C. The cell pellet was
thawed and resuspended in lysis buffer (50 mM
sodium phosphate, pH 8.0 containing 300 mM
NaCl, 10 mM imidazole) supplemented with
DNaseI (0.1 µg/mL), lysozyme (2 mg/ml) and
EDTA-free protease inhibitors cocktail (Roche).
Resuspended cells were disrupted by sonication
and cellular debris was removed from the lysate by
centrifugation at 16000 rpm for 50 min. Sfp was
purified by nickel affinity chromatography. After
an extensive wash with 50 mM sodium phosphate,
pH 8.0 containing 300 mM NaCl and 10 mM
imidazole buffer, the bound protein was eluted
with a linear imidazole gradient (30-250 mM).
Pure fractions, as determined by SDS-PAGE, were
pooled together for buffer-exchange into 50 mM
sodium phosphate, pH 8.0 containing 150 mM
NaCl, 5 mM DTT, 10 mM MgCl2 and 10 %
glycerol. Sfp was concentrated using an Amicon
pressure concentrator with a 10 K Dalton filter to
250 µM.
Phosphopantetheinylation of MbtB-ACP by Sfp.
To convert the apo form of MbtB-ACP to the
phosphopantetheinylated form, holo-MbtB-ACP,
the phosphopantetheinyl transferase Sfp was used.
100 µM Apo-MbtB-ACP was incubated with 0.2
µM Sfp in 50 mM Tris, pH 7.8 with 150 mM
NaCl, 1 mM CoASH and 2 mM DTT. The
reaction was allowed to proceed at 25 °C for 18
hrs before buffer-exchange into 50 mM Tris, pH
8.0, 150 mM NaCl. Traces of Sfp were removed
during MbtB-ACP concentration using two
successive Amicon filtrations, 30 K Dalton and 3
K Dalton cut-off filters respectively.
Measurement of Enzymatic Activity. The
enzymatic activity of MbtA was determined
spectrophotometrically by coupling the formation
of AMP to the reactions of myokinase, pyruvate
kinase and lactate dehydrogenase as described
previously (42). Reactions were performed in 100
mM Hepes pH 7.5, 10 mM MgCl2, 250 mM NaCl,
1 mM PEP, 0.15 mM NADH, 300 µM holo-MbtB-
ACP, 18 units of myokinase, 18 units of pyruvate
kinase, and 18 units of lactate dehydrogenase in a
final volume of 100 µL. Typically 0.5 µM of
Msmeg or Mtb MbtA was used and incubated for 5
min at 25 ºC with the reaction mix prior to
reaction initiation by substrate addition with 2,3-
dihydroxybenzoate, a salicylic acid analogue. The
reaction was monitored at 340 nm (ε340 = 6220 M-
1cm-1) using a Shimadzu spectrophotometer (UV-
2450).
Site-Directed Mutagenesis and Purification of
MbtA-K546A. The K546A mutation was
introduced into the pET-28a (+)::MbtA N-
terminally His6 tag plasmid using the Quickchange
mutagenesis kit (Stratagene) with the following
primers Mtb_MbtA_K542A_F (5’-
CAACGCCGATCGGGGCGATCGACAAACGA
G-3’), Mtb_MbtA_K542A_R (5’-
CTCGTTTGTCGATCGCCCCGATCGGCGTTG-
3’), Msmeg_MbtA_K546A_F (5’-
CACGGCCGTCGGCGCGATCGACAAGAAG-
3’) and Msmeg_MbtA_K546A_R (5’-
CTTCTTGTCGATCGCGCCGACGGCCGTG-
3’). The mutation was confirmed by DNA
sequencing. The expression and purification of
MbtA mutant was the same as described for the
wild type.
In vitro Acetylation Assay. 10 µM MbtA or the
mutant protein were incubated with 1 mM cAMP,
100 µM AcCoA and 1 µM Msmeg Pat at 37 °C for
5 hours. Samples were then analyzed by SDS-
PAGE. Proteins were transferred to a
nitrocellulose membrane. Western blots were
performed using an anti-acetyllysine antibody
(Cell Signaling Technology Inc. and
ImmuneChem Pharmaceuticals Inc., dilution of
1:2000) and Goat Anti-Rabbit IgG AP conjugate
(Bio-Rad Laboratories, dilution of 1:2000).
Development was carried out according to the
manufacturer’s instructions.
Time-Dependent Inactivation and reactivation of
MbtA. 20 µM MbtA was incubated with 1 mM
cAMP and 100 µM AcCoA with 5 µM Msmeg Pat
in 50 mM HEPES pH 7.5, 100 mM NaCl at 37 °C.
Aliquots of the reaction mixture were withdrawn
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
9!
every hour. The residual activity of MbtA was
measured as described above. Control experiments
were performed when Msmeg Pat or AcCoA were
not included. MbtA acetylation was also assessed
by western-blot. For reactivation time course, the
acetylated MbtA was then purified by Ni-NTA
chromatography and used in the deacetylation
assay. A typical reaction mixture contained 10 µM
acetylated MbtA, 2 mM NAD+, and 5 µM
Rv1151c (deacetylase). Aliquots of the reaction
mixture were withdrawn every hour and MbtA
activity was measured as described above. Control
experiments were performed without adding
NAD+ or the deacetylase. MbtA deacetylation was
also assessed by western-blot.
Mass Spectrometric Analysis of Acetylated MbtA.
The mass spectrometric analysis was performed at
the Laboratory for Macromolecular Analysis and
Proteomics (LMAP) of Albert Einstein College of
Medicine. The same protocol as described in
(19,43) was implemented for identification of
MbtA acetylation sites.
Media and Bacterial Cultures. Our growth studies
utilized a BSL2-safe derivative strain of Mtb
H37Rv called mc26206 (H37Rv ΔpanCD
ΔleuCD), which is routinely used by the Mtb
community (44). mc26206 is a strain auxotrophic
for both pantothenate and leucine (pan- leu
-) and
was maintained in Middlebrook 7H9 broth (Difco)
supplemented with 10 % ADC (5 % bovine serum
albumin [BSA], 2 % dextrose, 5 % catalase) 0.025
% tyloxapol, 50 µg/ml of pantothenate and 50
µg/ml of leucine. Antibiotics (hygromycin at 50
µg/ml and kanamycin at 25 µg/ml) were included
as appropriate for strains harboring antibiotic
resistance cassettes.
For growth in iron-limited medium,
mc26206 pan- leu
- strains were grown in
deferreted-Sauton. Sauton contains 0.2 % citric
acid, 0.05 % K2HPO4, 0.4 % L-asparagine, 0.2 %
dextrose, 6 % glycerol, 0.025 % tyloxapol, 50
µg/ml of pantothenate and 50 µg/ml of leucine. To
remove metal contamination, Sauton medium was
treated with Chelex-100 (Biorad) according to
manufacturer’s instructions. Chelex was removed
by filtration and the medium was supplemented
with 1 mg/L ZnSO4, 0.5 g/L MgSO4 and 16.2
mg/L ferric chloride. Iron limited Sauton medium
was supplemented with ZnSO4, MgSO4 as
described above and 0.162 mg/L of ferric chloride.
When required, kanamycin (25 µg/ml) and
hygromycin (50 µg/ml) were added to the
medium. Sauton, 7H9 and Sauton iron-limited
medium pHs were adjusted to 7.0 or 6.0 and 100
mM MES was added to maintain a constant pH
during growth curve.
Rv0998 and Rv1151c mutant constructions in Mtb.
The gene knockouts of Pat acetyltransferase
(Rv0998) and Sirtuin-like deacetylase DAc
(Rv1151c) were created by specialized
transduction methodology in mc26206 (44,45).
Constructs for allelic exchange were generated by
amplifying the upstream and downstream flanking
regions of each of the two genes using the primer
pairs listed in Table 2. The upstream and
downstream flanking regions were cloned into
suicidal delivery vector pYUB1471 to create an
allelic exchange vectors harboring a
selectable/counterselectable cassette [γδ(sacB-
Hyg)γδ] between the two flanking regions. The
allelic exchange constructs were incorporated into
shuttle mycobacteriophage vector phAE159; the
phasmid constructs used in this work were
obtained as part of a collaboration with Genomics
Institute of the Novartis Research Foundation
(GNF) to generate a set of gene deletion constructs
for Mtb (46). Phasmid DNA was electroporated
into Msmeg to obtain plaques at the permissive
temperature of 30 °C. Specialized transducing
phages were picked and amplified at 30 °C to
generate high titer mycobacteriophage. mc26206
was transduced with high-titer phages at the non-
permissive temperature of 37 °C to delete genes of
interest by specialized transduction (44,45). The
transductants were plated on selective medium:
Middlebrook 7H10 medium (Difco) containing
10% OADC enrichment (0.5 g oleic acid, 50 g
albumin, 20 g dextrose, 0.04 g catalase, 8.5 g
sodium chloride in 1 L water), 0.5% glycerol, 50
µg/ml leucine, 24 µg/ml pantothenate and 50
µg/ml hygromycin. Genomic DNA prepared from
transductants was screened by a three-primer PCR
to confirm gene deletion, using primers in Table 3.
For complementation, the Rv0998 and
Rv1151c genes were amplified by PCR from
H37Rv genomic DNA using the following primers
Rv00998_F
(TTTTTTTTGGATCCATTGGACGGGATAGC
CGAATTG), Rv0998_R
(TTTTTTTTGTTAACTCAGCCGACGGCCTCG
ATCAC), Rv1151c_F
(TTTTTTTTGGATCCAATGCGAGTGGCGGT
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
10!
GCTCAG), and Rv1151c_R
(TTTTTTTTGTTAACCTATTTCAGCAGGGCG
GGCAG) containing BamHI and HpaI
respectively. PCR products were digested with
BamHI and HpaI and cloned into the
mycobacterial episomal pMV261 vector using the
BamHI and HpaI restriction sites (47). After
sequence verification, complementation plasmids
were electroporated into the relevant deletion
mutant strains, with transformants selected on
7H10 medium as noted above, also containing 25
mg/ml kanamycin. To generate control strains for
comparison in growth studies, the pMV261 empty
vector was similarly electroporated into mc26206
parental, mc26206Pat and mc26206DAc.
Growth assays. Mycobacterial strains were
inoculated in liquid media, 7H9 pH 7 and then
transfered into fresh 7H9, Sauton or low-iron
Sauton media at either pH 7 or pH 6. After one
culture passage in the final medium to allow strain
adaptation, growth curves were started out at an
OD600 = 0.05 and optical densities were measured
at regular intervals for 20-40 days.
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
11!
REFERENCES
1. McDonough, W. F., and Sun, S. S. (1995) Composition of the Earth. Chemical Geology 120,
223-253
2. Raymond, K. N., and Carrano, C. J. (1979) Coordination chemistry and microbial iron transport.
Accounts of Chemical Research 12, 183-190
3. Posey, J. E., and Gherardini, F. C. (2000) Lack of a role for iron in the Lyme disease pathogen.
Science 288, 1651-1653
4. Raymond, K. N., Dertz, E. A., and Kim, S. S. (2003) Enterobactin: an archetype for microbial
iron transport. Proc Natl Acad Sci U S A 100, 3584-3588
5. Byrd, T. F., and Horwitz, M. A. (1993) Regulation of transferrin receptor expression and ferritin
content in human mononuclear phagocytes. Coordinate upregulation by iron transferrin and
downregulation by interferon gamma. J Clin Invest 91, 969-976
6. Comas, I., Coscolla, M., Luo, T., Borrell, S., Holt, K. E., Kato-Maeda, M., Parkhill, J., Malla, B.,
Berg, S., Thwaites, G., Yeboah-Manu, D., Bothamley, G., Mei, J., Wei, L., Bentley, S., Harris, S.
R., Niemann, S., Diel, R., Aseffa, A., Gao, Q., Young, D., and Gagneux, S. (2013) Out-of-Africa
migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat
Genet 45, 1176-1182
7. Jones, C. M., and Niederweis, M. (2011) Mycobacterium tuberculosis can utilize heme as an iron
source. J Bacteriol 193, 1767-1770
8. Tullius, M. V., Harmston, C. A., Owens, C. P., Chim, N., Morse, R. P., McMath, L. M., Iniguez,
A., Kimmey, J. M., Sawaya, M. R., Whitelegge, J. P., Horwitz, M. A., and Goulding, C. W.
(2011) Discovery and characterization of a unique mycobacterial heme acquisition system. Proc
Natl Acad Sci U S A 108, 5051-5056
9. De Voss, J. J., Rutter, K., Schroeder, B. G., Su, H., Zhu, Y., and Barry, C. E., 3rd. (2000) The
salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for
growth in macrophages. Proc Natl Acad Sci U S A 97, 1252-1257
10. Gobin, J., and Horwitz, M. A. (1996) Exochelins of Mycobacterium tuberculosis remove iron
from human iron-binding proteins and donate iron to mycobactins in the M. tuberculosis cell
wall. J Exp Med 183, 1527-1532
11. Ryndak, M. B., Wang, S., Smith, I., and Rodriguez, G. M. (2010) The Mycobacterium
tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain. J Bacteriol 192,
861-869
12. Quadri, L. E., Sello, J., Keating, T. A., Weinreb, P. H., and Walsh, C. T. (1998) Identification of a
Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the
virulence-conferring siderophore mycobactin. Chem Biol 5, 631-645
13. Luo, M., Fadeev, E. A., and Groves, J. T. (2005) Mycobactin-mediated iron acquisition within
macrophages. Nat Chem Biol 1, 149-153
14. Wells, R. M., Jones, C. M., Xi, Z., Speer, A., Danilchanka, O., Doornbos, K. S., Sun, P., Wu, F.,
Tian, C., and Niederweis, M. (2013) Discovery of a siderophore export system essential for
virulence of Mycobacterium tuberculosis. PLoS Pathog 9, e1003120
15. Chavadi, S. S., Stirrett, K. L., Edupuganti, U. R., Vergnolle, O., Sadhanandan, G., Marchiano, E.,
Martin, C., Qiu, W. G., Soll, C. E., and Quadri, L. E. (2011) Mutational and phylogenetic
analyses of the mycobacterial mbt gene cluster. J Bacteriol 193, 5905-5913
16. Krithika, R., Marathe, U., Saxena, P., Ansari, M. Z., Mohanty, D., and Gokhale, R. S. (2006) A
genetic locus required for iron acquisition in Mycobacterium tuberculosis. Proc Natl Acad Sci U
S A 103, 2069-2074
17. Rodriguez, G. M. (2006) Control of iron metabolism in Mycobacterium tuberculosis. Trends
Microbiol 14, 320-327
18. Pandey, R., and Rodriguez, G. M. (2014) IdeR is required for iron homeostasis and virulence in
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
12!
Mycobacterium tuberculosis. Mol Microbiol 91, 98-109
19. Vergnolle, O., Xu, H., and Blanchard, J. S. (2013) Mechanism and Regulation of Mycobactin
Fatty Acyl-AMP Ligase FadD33. J Biol Chem 288, 28116-28125
20. Lee, H. J., Lang, P. T., Fortune, S. M., Sassetti, C. M., and Alber, T. (2012) Cyclic AMP
regulation of protein lysine acetylation in Mycobacterium tuberculosis. Nat Struct Mol Biol 19,
811-818
21. Favrot, L., Blanchard, J. S., and Vergnolle, O. (2016) Bacterial GCN5-Related N-
Acetyltransferases: From Resistance to Regulation. Biochemistry 55, 989-1002
22. McDonough, K. A., and Rodriguez, A. (2012) The myriad roles of cyclic AMP in microbial
pathogens: from signal to sword. Nat Rev Microbiol 10, 27-38
23. Xu, H., Hegde, S. S., and Blanchard, J. S. (2011) Reversible Acetylation and Inactivation of
Mycobacterium tuberculosis Acetyl-CoA Synthetase Is Dependent on cAMP. Biochemistry 50,
5883-5892
24. May, J. J., Kessler, N., Marahiel, M. A., and Stubbs, M. T. (2002) Crystal structure of DhbE, an
archetype for aryl acid activating domains of modular nonribosomal peptide synthetases. Proc
Natl Acad Sci U S A 99, 12120-12125
25. Gulick, A. M. (2009) Conformational dynamics in the Acyl-CoA synthetases, adenylation
domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem Biol 4, 811-827
26. Drake, E. J., Duckworth, B. P., Neres, J., Aldrich, C. C., and Gulick, A. M. (2010) Biochemical
and structural characterization of bisubstrate inhibitors of BasE, the self-standing nonribosomal
peptide synthetase adenylate-forming enzyme of acinetobactin synthesis. Biochemistry 49, 9292-
9305
27. Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C.
(2003) The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-
propylphosphate and coenzyme A. Biochemistry 42, 2866-2873
28. Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997) Structural basis for the
activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. The EMBO
journal 16, 4174-4183
29. Reger, A. S., Carney, J. M., and Gulick, A. M. (2007) Biochemical and crystallographic analysis
of substrate binding and conformational changes in acetyl-CoA synthetase. Biochemistry 46,
6536-6546
30. Schmitt, M. P., Predich, M., Doukhan, L., Smith, I., and Holmes, R. K. (1995) Characterization
of an iron-dependent regulatory protein (IdeR) of Mycobacterium tuberculosis as a functional
homolog of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae. Infect
Immun 63, 4284-4289
31. Rodriguez, G. M., Voskuil, M. I., Gold, B., Schoolnik, G. K., and Smith, I. (2002) ideR, An
essential gene in mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression,
iron metabolism, and oxidative stress response. Infect Immun 70, 3371-3381
32. Pandey, S. D., Choudhury, M., Yousuf, S., Wheeler, P. R., Gordon, S. V., Ranjan, A., and
Sritharan, M. (2014) Iron-regulated protein HupB of Mycobacterium tuberculosis positively
regulates siderophore biosynthesis and is essential for growth in macrophages. J Bacteriol 196,
1853-1865
33. Ferreras, J. A., Ryu, J. S., Di Lello, F., Tan, D. S., and Quadri, L. E. (2005) Small-molecule
inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis. Nat
Chem Biol 1, 29-32
34. Nelson, K. M., Viswanathan, K., Dawadi, S., Duckworth, B. P., Boshoff, H. I., Barry, C. E., 3rd,
and Aldrich, C. C. (2015) Synthesis and Pharmacokinetic Evaluation of Siderophore Biosynthesis
Inhibitors for Mycobacterium tuberculosis. J Med Chem 58, 5459-5475
35. Lun, S., Guo, H., Adamson, J., Cisar, J. S., Davis, T. D., Chavadi, S. S., Warren, J. D., Quadri, L.
E., Tan, D. S., and Bishai, W. R. (2013) Pharmacokinetic and in vivo efficacy studies of the
mycobactin biosynthesis inhibitor salicyl-AMS in mice. Antimicrob Agents Chemother 57, 5138-
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
13!
5140
36. Vilcheze, C., Hartman, T., Weinrick, B., and Jacobs, W. R., Jr. (2013) Mycobacterium
tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction. Nat
Commun 4, 1881
37. Kabsch, W. (2010) Xds. Acta crystallographica. Section D, Biological crystallography 66, 125-
132
38. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R.
J. (2007) Phaser crystallographic software. Journal of applied crystallography 40, 658-674
39. Terwilliger, T. C., Grosse-Kunstleve, R. W., Afonine, P. V., Moriarty, N. W., Zwart, P. H., Hung,
L. W., Read, R. J., and Adams, P. D. (2008) Iterative model building, structure refinement and
density modification with the PHENIX AutoBuild wizard. Acta crystallographica. Section D,
Biological crystallography 64, 61-69
40. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of
Coot. Acta crystallographica. Section D, Biological crystallography 66, 486-501
41. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J.,
Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R.,
Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010)
PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta
crystallographica. Section D, Biological crystallography 66, 213-221
42. Pfleiderer, G., Kreiling, A., and Wieland, T. (1960) [On pantothenic acid synthetase from E. coli.
II. Quantitative enzymatic microdetermination of pantoyl acid, beta-alanine and pantothenic
acid]. Biochem Z 333, 308-310
43. Zheng, R., and Blanchard, J. S. (2001) Steady-State and Pre-Steady-State Kinetic Analysis of
Mycobacterium tuberculosis Pantothenate Synthetase. Biochemistry 40, 12904-12912
44. Jain, P., Hsu, T., Arai, M., Biermann, K., Thaler, D. S., Nguyen, A., Gonzalez, P. A., Tufariello,
J. M., Kriakov, J., Chen, B., Larsen, M. H., and Jacobs, W. R., Jr. (2014) Specialized transduction
designed for precise high-throughput unmarked deletions in Mycobacterium tuberculosis. MBio
5, e01245-01214
45. Bardarov, S., Bardarov Jr, S., Jr., Pavelka Jr, M. S., Jr., Sambandamurthy, V., Larsen, M.,
Tufariello, J., Chan, J., Hatfull, G., and Jacobs Jr, W. R., Jr. (2002) Specialized transduction: an
efficient method for generating marked and unmarked targeted gene disruptions in
Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148, 3007-3017
46. Tufariello, J. M., Malek, A. A., Vilcheze, C., Cole, L. E., Ratner, H. K., Gonzalez, P. A., Jain, P.,
Hatfull, G. F., Larsen, M. H., and Jacobs, W. R., Jr. (2014) Enhanced specialized transduction
using recombineering in Mycobacterium tuberculosis. MBio 5, e01179-01114
47. Stover, C. K., de la Cruz, V. F., Fuerst, T. R., Burlein, J. E., Benson, L. A., Bennett, L. T.,
Bansal, G. P., Young, J. F., Lee, M. H., Hatfull, G. F., and et al. (1991) New use of BCG for
recombinant vaccines. Nature 351, 456-460
AUTHOR CONTRIBUTIONS
OV, HX, JMT, JSB and WRJ planned and designed research. OV purified and LF crystallized MbtA
protein and determined its X-ray structure. OV and HX performed the in vitro enzyme activity. JMT
designed mutant constructs and mutants were obtained by HX and OV. OV, HX and AAM analyzed the
mutant phenotypes. OV, LF and JSB wrote the manuscript. All authors reviewed the results and approved
the final version of the manuscript.
The authors declare that they have no conflicts of interest with the contents of this article
ACKNOWLEDGEMENTS
We are grateful to Dr. Myrasol Callaway (Albert Einstein College of Medicine) for assistance in mass
spectrometry. We would like to thank Dr. Steven Almo and his laboratory for the use of their
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
14!
crystallography equipment.
FOOTNOTES
*This work was supported by NIH grants AI60899 (to J.S.B), AI26170, AI098925 and P01AI63537 (to
W.R.J).
**This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE)
Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory
under Contract No. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access
Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly
Company, which operates the facility.
FIGURE LEGENDS
Figure 1. Salicyl-capped mycobactin siderophore structure, genetic loci and first biosynthesis
reaction. A, lipophilic mycobactin (MBT) and hydrophilic carboxymycobactin (cMBT) share a common
core structure but differ in the length of the alkyl substitution (R group). Atoms in blue are involved in the
hexadentate ferric iron coordination. B, mbt-1 gene cluster produces mycobactin core whereas mbt-2 gene
cluster assembles and loads acyl fatty acid onto mycobactin lysine core. The black boxes indicate the
presence of IdeR binding sequences, which cause genes repression upon iron binding on IdeR. C,
adenylation-ligation reaction catalyzed by MbtA. ACP : acyl carrier protein domain of MbtB.
Figure 2. Pat acetylates specifically MbtA on Lys-546. A, Partial alignments of MbtA proteins from
M.smegmatis (MSMEG_4516) and M. tuberculosis (Rv2384) versus M. smegmatis FadD33
(MSMEG_2132). Acetylated lysines are indicated in bold. Conserved residues and basic residues
flanking acetylated sites are underlined. B, wild type MbtA or MbtA K546A mutant was incubated with
multiple reaction components as indicated in the table. The samples were analyzed by western-blot
(bottom panel) with acetyl-lysine antibody (a-AcK) and total protein content was determined by Ponceau
red (top panel). C, MbtA unique acetylation site was identified by MS/MS as Lys-546. Shown in an
MS/MS spectrum charged tryptic peptide from Ms MbtA (TTAVGK
AcIDKK) bearing an acetylated
lysine. The acetylated lysine is indicated as KAc.
Figure 3. Acetylation inhibits and deacetylation enhances the MbtA enzyme activity. A, time-
dependent inactivation of Msmeg MbtA by acetylation. Msmeg MbtA activity was monitored at different
time intervals with the additional components: (): cAMP, Msmeg Pat and acetyl-CoA; (): cAMP and
acetyl-CoA; (): cAMP and Msmeg Pat. B, time-dependent reactivation of acetylated Mtb MbtA by
deacetylation. After acetylation by Msmeg Pat, Mtb MbtA was then incubated with the following
components: (): NAD+ and Mtb DAc; ():Mtb DAc; (): NAD+.
Figure 4. Mtb DAc (Rv1151c) enzyme deacetylates both MbtA homologues. A, acetylated Mtb MbtA
were analyzed by western-blot (bottom panel) with acetyl-lysine antibody (α-AcK) and total protein
content was determined by Ponceau red (top panel). B, acetylated Msmeg MbtA were analyzed by
western-blot (bottom panel) with acetyl-lysine antibody (α-AcK) and total protein content was determined
by Ponceau red (top panel).
Figure 5. Crystal structure of apo Msmeg MbtA. A, Stereo view of the overall structure of apo Msmeg
MbtA. The three subdomains parts of the N-terminal domain are colored in purple (a), blue (b) and cyan
(c), respectively, while the C-terminal domain, or “lid”, is represented in orange. The location of the
active site is highlighted by a star . The residue K546, demonstrated in this manuscript to be acetylated, is
rendered as sticks colored by CPK, with carbon atoms in orange. B, Superimposition of the apo Msmeg
MbtA (green), B. subtilis DhbE in complex with DHB adenylated (blue, pdb accession code: 1MDB) and
Acinetobacter baumannii BasE N-terminal domain (yellow, pdb accession code: 3O83) structures. The
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
15!
DHB-adenylate product is displayed as sticks colored by CPK, with carbon atoms in magenta. C,
Superimposition of the apo Msmeg MbtA (green) and Salmonella enterica acetyl-CoA synthetase in
complex with CoA and AMP (orange, pdb accession code 2P2F) structures. The CoA and AMP ligands
are rendered as sticks colored by CPK, with carbon atoms in white.
Figure 6. Construction and verification of Mtb Pat (Rv0998) and Mtb DAc (Rv1151c) deletion
mutants. A and C, Rv0998 and Rv1151c adjacent gene organisations are shown. B and C, the ORF
removal in ΔRv0998 and ΔRv1151c deletion strains are replaced by sacB-hygR cassette. E and F, left and
right flanking PCR were used to confirm deletion of Rv0998 and Rv1151c with specific set of primers.
Figure 7. Iron level and pH effects on Mtb Pat and Mtb DAc deletion mutant phenotypes. Four
growth conditions are presented: A, Sauton pH7 medium; B, Sauton pH6 medium; C, limited iron Sauton
pH7 medium and D, limited iron Sauton pH6 medium for five constructs. (black), Mtb wild type +
pMV261; (solid red), Mtb Pat + pMV261; (dotted red), Mtb DAc + pMV261; (solid green), Mtb Pat
+ pMV261-Pat and (dotted green), Mtb DAc + pMV261-DAc. Error bars represent standard deviations
from the mean of results from biological duplicates. Mtb wild type corresponds to mc26206.
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
16!
TABLES
Table 1. Data collection and refinement statistics.
Data Collection
PDB ID
Space Group
Unit Cell Dimensions
a, b, c (Å)
α, β, γ (°)
Resolution Range (Å)
Wavelength (Å)
Rmerge (%) (Highest
shell)
CC(1/2) (Highest shell)
I/σI (Highest shell)
Completeness (%)
(Highest shell)
Multiplicity (Highest
shell)
Total Reflections
(Unique)
Refinement statistics
Rwork /Rfree
Number of non-
hydrogen atoms
Protein
Water
Average B-factors (Å2)
Protein
Water
Wilson B factor 2)
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
Ramachandran plot
Favored (%)
Outliers (%)
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
17!
Table 2. PCR primers for cloning flanks of the indicated loci, to generate allelic exchange
substrates.
Locus
Flank
Primer
name
Primer sequence
Rv1151c
Left
DRv1151c
LL
TTTTTTTTCCATAAATTGGGCCGCGGACCTGGTAAATAA
Left
DRv1151c
LR
TTTTTTTTCCATTTCTTGGTACAAGAATTGCAGGCTGTG-
GATGTCTCACTGAGGTCTCTTCTTGTCATCGCGGAACGTC
Right
DRv1151c
RR
TTTTTTTTCCATCTTTTGGCCAAGATCCATGCCCTGACC
Right
DRv1151c
RL
TTTTTTTTCCATAGATTGGCTGGTCTAGTAGTGTATAGC-
CGAGTGTCTGGTCTCGTAGGACGATCAGCATCCGCGAGT
Rv0998
Left
DRv0998
LL
TTTTTTTTCCATAAATTGGACGAAGGCATTCCGGTCAAA
Left
DRv0998
LR
TTTTTTTTCCATTTCTTGGAATCGTATGACACGCGCTTGG-
ATGTCTCACTGAGGTCTCTCCGGACATCCCTGAAAGACG
Right
DRv0998
RR
TTTTTTTTCCATCTTTTGGAGCTGCGTCCCGGAAAAAGT
Right
DRv0998
RL
TTTTTTTTCCATAGATTGGTTAATGATTGTGGCTGACGCC-
GAGTGTCTGGTCTCGTAGGTCCGGGTGAGCTGAGCTTG
Table 3. PCR Primers used to screen for deletions at the indicated loci.
Locus
Flank
Primer name
Binding site
Primer sequence
Rv1151c
Left
1151c L Flank
F=P7
Upstream of L flank, in
wild-type and mutant
CAT GCC GTT CAG
CAT GTC
sacBout_LR=P10
Within AES construct,
mutant only
GAT GTC TCA CTG
AGG TCT CT
1151c L Flank
R= P8
Within deleted region,
wild-type only
GGT GAT GAC GCT
GAC CTC
Right
1151c R Flank
F=P5
Within deleted region,
wild-type only
CGT CAT CAC CCA
GAA TGT C
hygout_RR= P9
Within AES construct,
mutant only
CGA GTG TCT GGT
CTC GTA G
1151c R Flank
R=P6
Downstream of R flank,
in wild-type and mutant
GGC ACT GTC GGA
TTA CAA G
Rv0998
Left
0998 L Flank
F=P1
Upstream of L flank, in
wild-type and mutant
CGT TGT GTC TAC
TGC TCG AC
sacBout_LR=P9
Within AES construct,
mutant only
GAT GTC TCA CTG
AGG TCT CT
0998 L Flank
R=P2
Within deleted region,
wild-type only
GAT GAT CGC AAC
ACC ATC
Right
0998 R Flank
F=P3
Within deleted region,
wild-type only
CGG TTC ATG TCG
GCT CGT GTT C
hygout_RR=P10
Within AES construct,
mutant only
CGA GTG TCT GGT
CTC GTA G
0998 R Flank
R=P4
Downstream of R flank,
in wild-type and mutant
TGT GCG GTA CAT
CGA CCA CCT C
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
18!
FIGURES
Figure 1.
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
19!
Figure 2.
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
20!
Figure 3
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
21!
Figure 4.
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
22!
Figure 5
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
23!
Figure 6
by guest on August 29, 2016http://www.jbc.org/Downloaded from
Mycobactin MbtA regulation!
!
24!
Figure 7
!
by guest on August 29, 2016http://www.jbc.org/Downloaded from
R. Jacobs, Jr. and John S. Blanchard
Olivia Vergnolle, Hua Xu, JoAnn M. Tufariello, Lorenza Favrot, Adel A. Malek, William
Biosynthesis
Post-Translational Acetylation of MbtA Modulates Mycobacterial Siderophore
published online August 26, 2016J. Biol. Chem.
10.1074/jbc.M116.744532Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted When this article is cited
to choose from all of JBC's e-mail alertsClick here
http://www.jbc.org/content/early/2016/08/26/jbc.M116.744532.full.html#ref-list-1
This article cites 0 references, 0 of which can be accessed free at
by guest on August 29, 2016http://www.jbc.org/Downloaded from
... 25 To carry out this two-step reaction, the Asub domain rotates by ~140° to adopt two unique conformations, namely the adenylate-forming conformation and a thioester-forming conformation. 22,26 Representative structures of stand-alone aryl adenylating enzymes have been solved where the Asub is present in the adenylation conformation, 27, 28 the thioester conformation in complex with the partner PCP domain, [29][30][31][32] and multiple intermediate conformations; 33 additionally, in the other structures, this dynamic subdomain is disordered and likely adopts multiple orientations within the crystal lattice. 34 In addition to exploring the reaction mechanism and domain conformations, studies e of substrate recognition and selectivity of aryl-adenylation domains have shown significant conservation of binding pocket residues in aryl acid binding adenylation domains found across different catechol siderophore biosynthetic pathways. ...
... 34 In addition to exploring the reaction mechanism and domain conformations, studies e of substrate recognition and selectivity of aryl-adenylation domains have shown significant conservation of binding pocket residues in aryl acid binding adenylation domains found across different catechol siderophore biosynthetic pathways. 27,30,[33][34][35][36][37][38] Structures of adenylation domains bound to non-native substrates also support efforts to understand the determinants of substrate promiscuity and to design novel inhibitors. 28,34 The substrate-recognizing residues in the binding pocket of the stand-alone adenylation domain EntE from E. coli show that Asn235 and Ser240 form hydrogen bonds with the two hydroxyl groups of DHB, whereas Tyr236, Val331, and Val339 form the base of a hydrophobic pocket for DHB. ...
Preprint
Full-text available
Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria Acinetobacter baumannii produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report on structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early-stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.
... The source organism was Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) with protein code P71716 (MBTA_MYCTU). The PDB-BLAST search outcomes demonstrated that Mtb-MbtA shares sequence identity with related proteins, such as the crystal structure of DhbE bound with 2,3-dihydroxybenzoic acid (DHB)-adenylate (PDB code 1MDB) from Bacillus subtilis and Mycobacterium smegmatis MbtA apo structure (PDB ID: 5KEI) [43,44]. The active site of the MbtA protein was identified by analogy with these homologous structures and was prepared using the protein tab in AutoDock 4.2.6 program by MGLTools 1.5.6 for docking. ...
Preprint
Full-text available
Tuberculosis (TB) continues to pose a global health challenge, exacerbated by the rise of drug-resistant strains. The development of new TB therapies is an arduous and time-consuming process. To expedite the discovery of effective treatments, computational structure-based drug re-purposing has emerged as a promising strategy. From this perspective, conditionally essential targets present a valuable opportunity, and the mycobactin biosynthesis pathway stands out as a prime example highlighting the intricate response of Mycobacterium tuberculosis (Mtb) to changes in iron availability. This study focuses on the re-purposing and revival of FDA-approved drugs (library) as potential inhibitors of MbtA, a crucial enzyme in mycobactin biosynthesis in Mtb conserved among all species of mycobacteria. Literature suggests this pathway to be associated with drug efflux pumps, which potentially contribute to drug resistance. This makes it a potential target for antitubercular drug discovery. Herein we utilized cheminformatics and structure-based drug repurposing approaches, viz., molecular docking, dynamics, and PCA analysis, to decode the intermolecular interactions and binding affinity of the FDA-reported molecules against MbtA. The virtual screening revealed ten molecules with significant binding affinities and interactions with MbtA. These drugs, originally designed for different therapeutic indications (4: antiviral, 3: anticancer, 1: CYP450 inhibitor, 1: ACE inhibitor, and 1: leukotriene antagonist), are repurposed as potential MbtA inhibitors. Furthermore, our study explores the binding modes and interactions between these drugs and MbtA, shedding light on the structural basis of their inhibitory potential. Principal component analysis highlighted significant motions in MbtA-bound ligands, emphasizing the stability of the top Protein-Ligand Complexes (PLCs). This computational approach provides a swift and cost-effective method for identifying new MbtA inhibitors, which can subsequently undergo validation through experimental assays. This streamlined process is facilitated by the fact that these compounds are already FDA-approved and have established safety and efficacy profiles. This study has the potential to lay the groundwork for addressing the urgent global health challenge at hand, specifically in the context of combating Antimicrobial Resistance (AMR) and Tuberculosis (TB).
... The source organism was Mycobacterium tuberculosis (strain ATCC 25618/H37Rv) with protein code P71716 (MBTA_MYCTU). The PDB-BLAST search outcomes demonstrated that Mtb-MbtA shares sequence identity with related proteins, such as the crystal structure of DhbE bound with 2,3-dihydroxybenzoic acid (DHB)-adenylate (PDB code 1MDB) from the Bacillus subtilis and Mycobacterium smegmatis MbtA apo structure (PDB ID: 5KEI) [43,44]. The active site of the MbtA protein was identified by analogy with these homologous structures and was prepared using the protein tab in the AutoDock 4.2.6 program by MGLTools 1.5.6 for docking. ...
Article
Full-text available
Tuberculosis (TB) continues to pose a global health challenge, exacerbated by the rise of drug-resistant strains. The development of new TB therapies is an arduous and time-consuming process. To expedite the discovery of effective treatments, computational structure-based drug repurposing has emerged as a promising strategy. From this perspective, conditionally essential targets present a valuable opportunity, and the mycobactin biosynthesis pathway stands out as a prime example highlighting the intricate response of Mycobacterium tuberculosis (Mtb) to changes in iron availability. This study focuses on the repurposing and revival of FDA-approved drugs (library) as potential inhibitors of MbtA, a crucial enzyme in mycobactin biosynthesis in Mtb conserved among all species of mycobacteria. The literature suggests this pathway to be associated with drug efflux pumps, which potentially contribute to drug resistance. This makes it a potential target for antitubercular drug discovery. Herein, we utilized cheminformatics and structure-based drug repurposing approaches, viz., molecular docking, dynamics, and PCA analysis, to decode the intermolecular interactions and binding affinity of the FDA-reported molecules against MbtA. Virtual screening revealed ten molecules with significant binding affinities and interactions with MbtA. These drugs, originally designed for different therapeutic indications (four antiviral, three anticancer, one CYP450 inhibitor, one ACE inhibitor, and one leukotriene antagonist), were repurposed as potential MbtA inhibitors. Furthermore, our study explores the binding modes and interactions between these drugs and MbtA, shedding light on the structural basis of their inhibitory potential. Principal component analysis highlighted significant motions in MbtA-bound ligands, emphasizing the stability of the top protein–ligand complexes (PLCs). This computational approach provides a swift and cost-effective method for identifying new MbtA inhibitors, which can subsequently undergo validation through experimental assays. This streamlined process is facilitated by the fact that these compounds are already FDA-approved and have established safety and efficacy profiles. This study has the potential to lay the groundwork for addressing the urgent global health challenge at hand, specifically in the context of combating antimicrobial resistance (AMR) and tuberculosis (TB).
Article
Full-text available
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), faces iron scarcity within the host due to immune defenses. This review explores the importance of iron for Mtb and its strategies to overcome iron restriction. We discuss how the host limits iron as an innate immune response and how Mtb utilizes various iron acquisition systems, particularly the siderophore-mediated pathway. The review delves into the structure and biosynthesis of mycobactin, a key siderophore in Mtb, and the regulation of its production. We explore the potential of targeting siderophore biosynthesis and uptake as a novel therapeutic approach for TB. Finally, we summarize current knowledge on Mtb's iron acquisition and highlight promising directions for future research to exploit this pathway for developing new TB interventions.
Article
Full-text available
This review highlights the utility of using adenylation domain structural data, biochemical assays, and computational predictions for prioritizing nonribosomal peptide pathways for natural product discovery.
Article
Lysine acetylation is a conserved regulatory post-translational protein modification that is performed by lysine acetyltransferases (KATs). By catalyzing the transfer of acetyl groups to substrate proteins, KATs play critical regulatory roles in all domains of life; however, no KATs have yet been identified in cyanobacteria. Here, we tested all predicted KATs in the cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) and demonstrated that A1596, which we named cyanobacterial Gcn5-related N-acetyltransferase (cGNAT2), can catalyze lysine acetylation in vivo and in vitro. Eight amino acid residues were identified as the key residues in the putative active site of cGNAT2, as indicated by structural simulation and site-directed mutagenesis. The loss of cGNAT2 altered both growth and photosynthetic electron transport in Syn7002. In addition, quantitative analysis of the lysine acetylome identified 548 endogenous substrates of cGNAT2 in Syn7002. We further demonstrated that cGNAT2 can acetylate NAD(P)H dehydrogenase J (NdhJ) in vivo and in vitro, with the inability to acetylate K89 residues, thus decreasing NdhJ activity and affecting both growth and electron transport in Syn7002. In summary, this study identified a KAT in cyanobacteria and revealed that cGNAT2 regulates growth and photosynthesis in Syn7002 through an acetylation-mediated mechanism.
Article
Full-text available
Covering: up to fall 2022.Nonribosomal peptide synthetases (NRPSs) are a family of modular, multidomain enzymes that catalyze the biosynthesis of important peptide natural products, including antibiotics, siderophores, and molecules with other biological activity. The NRPS architecture involves an assembly line strategy that tethers amino acid building blocks and the growing peptides to integrated carrier protein domains that migrate between different catalytic domains for peptide bond formation and other chemical modifications. Examination of the structures of individual domains and larger multidomain proteins has identified conserved conformational states within a single module that are adopted by NRPS modules to carry out a coordinated biosynthetic strategy that is shared by diverse systems. In contrast, interactions between modules are much more dynamic and do not yet suggest conserved conformational states between modules. Here we describe the structures of NRPS protein domains and modules and discuss the implications for future natural product discovery.
Article
Full-text available
Article
Full-text available
Unlabelled: Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis. Importance: This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.
Article
Full-text available
Unlabelled: G: enetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria. Importance: Genetic manipulation of M. tuberculosis is hampered by laborious and relatively inefficient methods for generating deletion mutant strains. The combined use of phage-based transduction and recombineering methods greatly enhances the efficiency by which knockout strains can be generated. The additional elimination of recD further enhances this efficiency. The methods described herein will facilitate the construction of comprehensive gene knockout libraries and expedite the isolation of previously difficult to recover mutants, promoting antimicrobial and vaccine development.
Article
Full-text available
Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe3+-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 μg Fe ml−1), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.
Article
Full-text available
Tuberculosis caused 20% of all human deaths in the Western world between the seventeenth and nineteenth centuries and remains a cause of high mortality in developing countries. In analogy to other crowd diseases, the origin of human tuberculosis has been associated with the Neolithic Demographic Transition, but recent studies point to a much earlier origin. We analyzed the whole genomes of 259 M. tuberculosis complex (MTBC) strains and used this data set to characterize global diversity and to reconstruct the evolutionary history of this pathogen. Coalescent analyses indicate that MTBC emerged about 70,000 years ago, accompanied migrations of anatomically modern humans out of Africa and expanded as a consequence of increases in human population density during the Neolithic period. This long coevolutionary history is consistent with MTBC displaying characteristics indicative of adaptation to both low and high host densities.
Article
Full-text available
Mycobacterial siderophores are critical components for bacterial virulence in the host. Pathogenic mycobacteria synthesize iron chelating siderophores named mycobactin and carboxymycobactin to extract intracellular macrophage iron. The two siderophores differ in structure only by a lipophilic aliphatic chain attached on the ϵ-amino group of the lysine mycobactin core, which is transferred by MbtK. Prior to acyl chain transfer, the lipophilic chain requires activation by a specific fatty acyl-AMP ligase FadD33 (also known as MbtM) and is then loaded onto phosphopantetheinylated acyl carrier protein (holo-MbtL) to form covalently acylated MbtL. We demonstrate that FadD33 prefers long chain saturated lipids and initial velocity studies showed that FadD33 proceeds via a Bi Uni Uni Bi ping-pong mechanism. Inhibition experiments suggest that, during the first half-reaction (adenylation), fatty acid binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), holo-MbtL binds to the enzyme followed by the release of products AMP and acylated MbtL. In addition, we characterized a post-translational regulation mechanism of FadD33 by the mycobacterial protein lysine acetyltransferase in a cAMP-dependent manner. FadD33 acetylation leads to enzyme inhibition, which can be reversed by the NAD⁺-dependent deacetylase, MSMEG_5175 (DAc1). To the best of our knowledge, this is the first time that bacterial siderophore synthesis has been shown to be regulated via post-translational protein acetylation. Background: Fatty acyl-AMP ligase FadD33 is required for mycobactin biosynthesis. Results: FadD33 catalyzes a two-step kinetic mechanism, and FadD33 activity is regulated by post-translational acetylation. Conclusion: Mycobactin FadD33 activity is reversibly regulated by Pat (acetylation) and DAc1 (deacetylation). Significant: Post-translational regulation via acetylation of enzymes can modulate siderophore biosynthesis.
Article
The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that include more than 100,000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-acetyltransferases transfer an acetyl group from acetyl-CoA (AcCoA) to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur on two different positions, either at the amino terminal end (αN-acetylation) or the epsilon-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups based on their substrate specificity and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics and their mechanism of action.
Article
MbtA catalyzes the first committed biosynthetic step of the mycobactins, which are important virulence factors associated with iron acquisition in Mycobacterium tuberculosis. MbtA is a validated therapeutic target for antitubercular drug development. 5'-O-[N-(salicyl)sulfamoyl]adenosine (1) is a bisubstrate inhibitor of MbtA and exhibits exceptionally potent biochemical and antitubercular activity. However, 1 suffers from sub-optimal drug disposition properties resulting in a short half-life (t1/2), low exposure (AUC), and low bioavailability (F). Four strategies were pursued to address these liabilities including the synthesis of prodrugs, increasing the pKa of the acyl-sulfonyl moiety, modulation of the lipophilicity, and strategic introduction of fluorine into 1. Complete pharmacokinetic (PK) analysis of all compounds was performed. The most successful modifications involved fluorination of the nucleoside that provided substantial improvements in t1/2 and AUC. Increasing the pKa of the acyl-sulfonyl linker yielded incremental enhancements while modulation of the lipophilicity and prodrug approaches led to substantially poorer PK parameters.
Article
The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of approximately 60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 A resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94 degrees occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the alpha-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.
Article
Iron is an essential but potentially harmful nutrient, poorly soluble in aerobic conditions, and not freely available in the human host. To acquire iron, bacteria have evolved high affinity iron acquisition systems that are expressed under iron limitation often in conjunction with virulence determinants. Because excess iron can be dangerous, intracellular iron must be tightly controlled. In mycobacteria, IdeR functions as a global iron dependent transcriptional regulator, but because inactivation of ideR is lethal for Mycobacterium tuberculosis, it has not been possible to use genetics to fully characterize this protein's function or examine the requirement of iron regulation during tuberculosis infection. In this work, a conditional M. tuberculosis ideR mutant was generated and used to study the basis of IdeR's essentiality. This investigation uncovered positive regulation of iron storage as a critical aspect of IdeR's function in regular culture and a prominent factor for survival under stresses associated with life in macrophages. Moreover, this study demonstrates that IdeR is indispensable in the mouse model of tuberculosis, thereby linking iron homeostasis to virulence in M. tuberculosis.