Biochem. J. (2014) 461, 87–98 (Printed in Great Britain) doi:10.

1042/BJ20140209 87

Alternative substrates reveal catalytic cycle and key binding events in the
reaction catalysed by anthranilate phosphoribosyltransferase from
Mycobacterium tuberculosis

www.biochemj.org
Tammie V. M. COOKSON*1 , Alina CASTELL†1,2 , Esther M. M. BULLOCH†, Genevieve L. EVANS†, Francesca L. SHORT†3 ,
Edward N. BAKER†, J. Shaun LOTT†4 and Emily J. PARKER*4
*Maurice Wilkins Centre for Molecular Biodiscovery, Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8140,
New Zealand
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand

AnPRT (anthranilate phosphoribosyltransferase), required for
the biosynthesis of tryptophan, is essential for the virulence
of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the
Mg2 + -dependent transfer of a phosphoribosyl group from
PRPP (5 -phosphoribosyl-1 -pyrophosphate) to anthranilate to
form PRA (5 -phosphoribosyl anthranilate). Mtb-AnPRT was
shown to catalyse a sequential reaction and significant substrate
inhibition by anthranilate was observed. Antimycobacterial
fluoroanthranilates and methyl-substituted analogues were shown
to act as alternative substrates for Mtb-AnPRT, producing
the corresponding substituted PRA products. Structures of the
enzyme complexed with anthranilate analogues reveal two distinct
binding sites for anthranilate. One site is located over 8 Å (1 Å =
0.1 nm) from PRPP at the entrance to a tunnel leading to the active
PRPP, in a catalytically relevant position. Soaking the analogues
for variable periods of time provides evidence for anthranilate
located at transient positions during transfer from the outer site to
the inner catalytic site. PRPP and Mg2 + binding have been shown
to be associated with the rearrangement of two flexible loops,
which is required to complete the inner anthranilate-binding site. It
is proposed that anthranilate first binds to the outer site, providing
an unusual mechanism for substrate capture and efficient transfer
to the catalytic site following the binding of PRPP.
Key words: anthranilate phosphoribosyltransferase (AnPRT),
fluoroanthranilate, Mycobacterium tuberculosis, trpD, tryptophan
biosynthesis, tuberculosis.

Biochemical Journal
site, whereas in the second, inner, site anthranilate is adjacent to

INTRODUCTION
The worldwide emergence of extensively drug-resistant
Mycobacterium tuberculosis (Mtb), the causative agent of TB
(tuberculosis) [1], has been suggested to be one of the most
profound challenges facing global health [2]. Treatment of TB
requires a multidrug approach [3] and thus there is a desperate
need for new and effective anti-TB drugs that function via novel
modes of action [4].
Recent studies have shown that host CD4 + T-cells employ
tryptophan starvation in an attempt to eradicate M. tuberculosis
infection [5]. This innate immune response is effective against
organisms that are natural tryptophan auxotrophs, but as
the tryptophan biosynthetic pathway can normally overcome
this starvation in M. tuberculosis, the host immune response
is rendered ineffective. Drugs that inhibit the tryptophan
biosynthetic pathway could therefore work synergistically with
the host immune response to starve M. tuberculosis of this
essential amino acid. A tryptophan auxotrophic mutant of
the virulent M. tuberculosis strain H37Rv, with a deletion
of the gene encoding the enzyme that catalyses the second
committed step in tryptophan biosynthesis, AnPRT (anthranilate
phosphoribosyltransferase) (also known as trpD; EC 2.4.2.18),
has a decreased ability to multiply in murine macrophages
and is essentially avirulent in both immunocompetent and
immunocompromised mice [6]. AnPRT, like the other enzymes
involved in tryptophan biosynthesis, is absent from mammals, so
inhibitors of this enzyme may have limited mammalian toxicity,
strengthening it further as an attractive target for anti-TB drug
design.
AnPRT is a member of the PRT (phosphoribosyltransferase)
family of enzymes characterized by their common substrate
PRPP (5 -phosphoribosyl-1 -pyrophosphate) [7], which acts as
the phosphoribosyl donor in a substitution reaction with a
nitrogenous, and generally aromatic, base [8]. At least four distinct
structural types of PRT have been identified, with AnPRT the sole
representative of a type III PRT [9]. Type I PRTs are the most
common, and enzymes in this class act on purines and pyrimidines
as part of salvage pathways (e.g. adenine PRT, hypoxanthine PRT
or uracil PRT) or in the case of orotate PRT as part of the de

Abbreviations: AnPRT, anthranilate phosphoribosyltransferase; Eco , Escherichia coli ; FA, fluoroanthranilate; F-PRA, fluoro-PRA; InGP, indole glycerol
phosphate; InGPS, indole glycerol phosphate synthase; KIE, kinetic isotope effect; MA, methylanthranilate; MESG, 2-amino-6-mercapto-7-methylpurine
ribonucleoside; M-PRA, methyl-PRA; Mtb , Mycobacterium tuberculosis ; NP, nucleoside phosphorylase; PNP, purine nucleoside phosphorylase; PR, 5 -
phosphoribosyl; PRA, 5 -phosphoribosyl anthranilate; PRAI, 5 -phosphoribosyl anthranilate isomerase; PRPP, 5 -phosphoribosyl-1 -pyrophosphate; PRT,
phosphoribosyltransferase; Sso , Sulfolobus solfataricus ; TB, tuberculosis.
1
These authors contributed equally to this work.
2
Present address: Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, 171 77 Stockholm, Sweden.
3
Present address: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
4
Correspondence may be addressed to either of these authors (email s.lott@auckland.ac.nz or emily.parker@canterbury.ac.nz).
The structural co-ordinates reported for Mycobacterium tuberculosis anthranilate phosphoribosyltransferase complexed with anthranilate or anthranilate
analogues have been deposited in the PDB under codes 4N5V, 4N8Q, 4N93, 4OWM, 4OWN, 4OWO, 4OWQ, 4OWS, 4OWU and 4OWV.


c The Authors Journal compilation 
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88 T. V. M. Cookson and others
novo pyrimidine biosynthetic pathway [9]. Quinolate PRT and
nicotinate PRT are type II PRTs, and ATP PRT, which catalyses
the first step of histidine biosynthesis, is the only type IV PRT
identified to date.
The displacement of PPi from PRPP by a nitrogenous base
that is catalysed by PRTs takes place with inversion of the
stereochemistry at C1 of the ribose ring. Reactions of this
type can be either SN 1-like, in which PPi release precedes
nucleophilic attack on PRPP by the base, generating a short-
lived oxocarbenium ion intermediate, or SN 2-like, where the
nucleophilic attack and PPi loss occur in the same reaction step.
KIE (kinetic isotope effect) experiments with type I orotate PRT
are consistent with a transition state that has extensive ribo-
oxocarbenium ion character [10–13]. Structures of type I PRT
enzymes in complex with substrates and transition state analogues
show that C1 of the ribosyl ring adopts various positions away
from PPi and towards the nitrogenous base, as would be consistent
with an SN 1 mechanism [9,14,15]. It has been proposed that
type I PRTs guide catalysis by binding PRPP in an orientation
that favours its breakdown into PR (5 -phosphoribosyl) and PPi
[16,17].
AnPRT catalyses the transfer of PR from PRPP to anthranilate.
AnPRT is structurally distinct from the other PRT types, and in-
stead has a similar structural fold, quaternary structure and active-
site location to that of type II NPs (nucleoside phosphorylases)
[18,19]. Type II NP enzymes catalyse glycosyl transfer reactions
that result in the production of ribose 1-phosphate, which is the
near-reverse reaction of that catalysed by AnPRT [18,19]. To date,
most of the NPs characterized using KIEs are of type I and have
been shown to have SN 1-like transition states. The only type II
NP characterized by KIEs, human thymidine phosphorylase, has
been proposed to have an SN 2 transition state [20,21]. Thus it is
not clear whether AnPRT catalyses an SN 1 or an SN 2 reaction
(Figure 1). Further evidence about the catalytic mechanism of
AnPRT will aid in inhibitor design.
We have previously determined crystal structures of Mtb-
AnPRT with and without PRPP (PDB codes 2BPQ, 1ZVW
and 3QR9) [22,23]. The enzyme crystallizes as a homodimer
with one active site in each subunit. Each monomer comprises
two domains: a smaller α-helical N-terminal domain, and a
larger C-terminal domain containing both α-helices and β-sheets.
A hinge region, consisting of the α4–β1, β3–α8 and α9–β4
connecting loops, links the two domains. Dimer formation occurs
through interactions between the helical N-terminal domains, as
is also observed for the AnPRT enzymes from Acinetobacter
baylyi (PDB code 4GTN), Sulfolobus solfataricus (Sso) [24],
Pectobacterium carotovorum (PDB codes 1KGZ and 1KHD)
[19], Thermus thermophilus (PDB codes 1V8G and 2ELC)
and Xanthomonas campestris (PDB code 4HKM). The PRPP
substrate has been shown to bind in the C-terminal domain
of the enzyme, at the bottom of a deep cleft, with the PPi
group buried the most deeply. When compared with the ligand-
free protein structure, two loops (the β1–α5 and β2–α6 loops)
can be identified in the PRPP- and Mg2 + -bound structure that
move out of the PRPP-binding site in order to accommodate
the PRPP substrate. Lee et al. [22] used in silico docking
to predict the binding site of the enzyme’s second substrate,
anthranilate, and surprisingly predicted two sites despite a
1:1 reaction stoichiometry with PRPP (Figure 1). Two similar
anthranilate-binding sites (S1 and S2) were observed directly
in Sso-AnPRT (PDB codes 1ZYK and 2GVQ) [25], and a
third anthranilate-binding site most distal to the PRPP (S3) has
been proposed to be part of a substrate capture mechanism that
was also utilized in the binding of Mtb-AnPRT inhibitors [23].
Structures of Mtb-AnPRT in complex with bi-anthranilate-like

c The Authors Journal compilation 
c 2014 Biochemical Society
inhibitors show binding across both S1 and S2 and S2 and S3
[23,26].
In the present study, we investigated the Mtb-AnPRT catalytic
mechanism using compounds that have been reported recently to
have antimycobacterial activity both in vitro and in a mouse model
of disease [5]. The present study reveals that Mtb-AnPRT exhibits
a broad substrate tolerance, and analysis of the crystal structures
of the enzyme in complex with these alternative substrates has
illuminated some details of the reaction mechanism.
EXPERIMENTAL
Materials
Unless otherwise stated, all chemicals were obtained from Sigma–
Aldrich. Anthranilate was obtained from Merck Schuchardt. 4-
Fluoroanthranilate was obtained from Sigma–Aldrich, whereas
3-, 5- and 6-fluoroanthranilate and 3- and 4-methylanthranilate
were obtained from Fluorochem.
Protein preparation
The Mtb-AnPRT protein was expressed and purified as described
previously [23]. The Escherichia coli (Eco) trpFC gene had been
cloned previously into the expression vector pProEX-HTb, which
adds an N-terminal His6 tag and rTEV (recombinant tobacco
etch virus) site. This plasmid was transformed into E. coli
BL21(DE3) cells containing plasmids (pBB528 and pBB541) for
co-expression of chaperonins (GroES and GroEL) [27].
The E. coli fusion protein of PRAI (5 -phosphoribosyl
anthranilate isomerase) and InGPS (indole glycerol phosphate
synthase) was purified as described for the Mtb-AnPRT protein
with the omission of the size-exclusion chromatography step. For
the NMR analyses, the proteins were dialysed against 150 mM
NH4 HCO3 (pH 8.0).
Protein concentrations were determined using a Nanodrop ND-
1000 spectrophotometer (Thermo Scientific) and molar absorp-
tion coefficient values of 34 615 and 40 715 litres · mol − 1 · cm − 1
for Mtb-AnPRT and Eco-PRAI–InGPS respectively.
Kinetics
A coupled assay where PRA (5 -phosphoribosyl anthranilate)
formation is detected by the formation of InGP (indole glycerol
phosphate) was used to determine the enzyme kinetic parameters
of Mtb-AnPRT as described previously [23].
The final concentrations of the following components were
included in the coupled assay: PRPP (0–65 μM), MgCl2 (1 mM),
Mtb-AnPRT (0.1 μM), Eco-PRAI–InGPS (1.7 μM), anthranilate
or anthranilate analogue (0–10 μM) and Tris buffer (50 mM
Tris/HCl and 150 mM NaCl, pH 8.0). Reactions were initiated
with the addition of Mtb-AnPRT, with initial rates of reaction
determined by least-squares fit of the initial rate data. Raw data
were fitted to the ternary mechanism equation:
Vmax [A] [B]
v=
K A K B + K B [A] + K A [B] + [A] [B]
using GraFit 5 (Erithacus Software).
The commercially available EnzChek® Pyrophosphate Assay
Kit (Invitrogen) was used to determine the kinetic parameters
pertaining to alternative substrate turnover, as not all compounds
were turned over by Eco-PRAI–InGPS. Along with PRA,
Mtb-AnPRT produces PPi in a 1:1 stoichiometric ratio with
 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase 89

Figure 1 The biosynthesis of tryptophan from anthranilate

Two possible mechanisms are shown for the reaction catalysed by AnPRT (shown in grey): an SN 1-like reaction passing through an oxocarbenium ion intermediate is depicted as the top route, and
an SN 2-like reaction where dissociation of PPi and addition of anthranilate take place in the same reaction step as the bottom route.

anthranilate. In this assay, the PPi produced by the Mtb-AnPRT anthranilate/anthranilate analogue (0–1 mM), MESG (200 μM),
reaction is converted first into Pi by inorganic pyrophosphatase PNP (1 unit), inorganic pyrophosphatase (0.03 unit) and reaction
(1:2), which is then reacted with the substrate MESG (2- buffer (50 mM Tris/HCl and 1 mM MgCl2 , pH 7.5, containing
amino-6-mercapto-7-methylpurine ribonucleoside), which has an 100 μM sodium azide) made up to 1 ml. Reactions were initiated
absorbance maximum of 330 nm, by PNP (purine nucleoside with the addition of Mtb-AnPRT, with initial rates of reaction
phosphorylase) in a 1:1 ratio. The products of this reaction determined by least-squares fit of the initial rate data. Apparent
are ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine; K m values were obtained by fitting the data to the Michaelis–
the latter can be monitored spectrophotometrically at 360 nm. Menten equation:
Inorganic pyrophosphatase, PNP and Eco-PRAI–InGPS were all
added in excess to ensure that the AnPRT-catalysed reaction was Vmax [S]
V =
the rate-limiting step. The molar absorption coefficient for 2- K m + [S]
amino-6-mercapto-7-methylpurine production from anthranilate
and PRPP was determined to be 26 200 litres · mol − 1 · cm − 1 under whereas substrate inhibition data were fitted to the substrate
assay conditions using known concentrations of anthranilate. inhibition equation:
All assays were measured with a Varian Cary 100 UV-VIS
Vmax [S]
spectrophotometer using quartz cuvettes thermally equilibrated V =  
to 22 ◦ C. The final concentrations of the following components K m + [S] 1 + [S]
Ki
were included in the coupled assay: PRPP (120 μM), MgCl2
(1 mM), Mtb-AnPRT (0.1 μM), Eco-PRAI–InGPS (6 μM), using GraFit 5.


c The Authors Journal compilation 
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90 T. V. M. Cookson and others
Mass spectroscopy
Solutions for determination of the mode of epimerization of PRA
via MS contained 2 mM PRPP, 2 mM anthranilate, 1 mM MgCl2
and 20 mM NaCNBH3 made up to 2 ml with Tris buffer. Solutions
were mixed by inversion and 0.2 μM Mtb-AnPRT was added.
After a 5 min reaction time, solutions were spun through Vivaspin
10 000 Da molecular-mass cut-off concentrators to remove the
enzyme present, flash-frozen with liquid nitrogen and left to freeze
dry (FreeZone 2.5, Labconco) for 4–5 days. Dried products were
then resuspended in ultrapure water and analysed via UHR-TOF
(ultra-high-resolution TOF) MS/MS (Bruker maXis 3G).
Solutions for analysis of Mtb-AnPRT/Eco-PRAI–InGPS
reaction products contained 1.2 mM PRPP, 400 μM an-
thranilate/fluoroanthranilate or 2 mM methylanthranilate, 2 mM
MgCl2 , 0.6 μM Mtb-AnPRT, 6 μM Eco-PRAI–InGPS made up
to 500 μl with ultrapure water for anthranilate/fluoroanthranilate
or 5 mM NH4 HCO3 for methylanthranilate. Reactions with
anthranilate or fluoroanthranilate were initiated with Mtb-AnPRT
immediately before analysis via MS, whereas reactions for
methylanthranilate were incubated with all substituents present
for 1 h before analysis.
NMR experiments
Assay solutions for analysis of Mtb-AnPRT-catalysed reactions
by 1 H-NMR spectroscopy contained 2–2.5 mM PRPP, 0.5–1 mM
anthranilate or anthranilate analogue, 0.2–0.8 μM Mtb-AnPRT,
2 mM MgCl2 , 0.25 mM trimethylsilyl propanoic acid, 80–90 %
(v/v) 2 H2 O and 10 mM NH4 HCO3 (pH 8). The 1D 1 H-NMR
experiments were acquired at 25 ◦ C on a DRX-400 spectrometer
(Bruker), or an AV-600 spectrometer (Bruker) fitted with a
cryoprobe, and using presaturation of the water resonance [28]. A
1D 1 H-NMR spectrum of each assay solution was collected before
addition of Mtb-AnPRT, and then after addition of the enzyme
at 100–300 s intervals, with averaging over 16–64 transients. In
experiments with Eco-PRAI–InGPS, this enzyme was added at
a concentration of 2.5 μM to an NMR sample in which PRA or
PRA analogue had been formed in situ using the assay conditions
detailed above, without removal of Mtb-AnPRT.
Crystallization
Crystallization of native Mtb-AnPRT has been described
previously [23]. For soaking experiments and data collection,
native crystals were removed from the crystallization drop with
a nylon loop. The native Mtb-AnPRT crystals were soaked
in a mixture of the mother liquor with 5 mM PRPP, 2.5 mM
MgCl2 and the alternative substrate to a final concentration
of 1 mM or anthranilate to a final concentration of 5 nM
for various periods of time (Supplementary Table S1 at
http://www.biochemj.org/bj/461/bj4610087add.htm). The soaked
crystals were quickly pulled through a drop of cryoprotectant
containing the mother liquor and 26 % (v/v) glycerol before being
flash-cooled in liquid nitrogen.
Data collection, structure solution and refinement
Data were collected at the Australian Synchrotron, Beamline
MX2, within 72 h. The crystals of the Mtb-AnPRT–anthranilate
analogue complexes diffracted to a maximum resolution that
varied between 1.89 and 2.43 Å (1 Å = 0.1 nm). X-ray diffraction
data were indexed, integrated and scaled with the autoPROC suite
[29], XDS [30] and SCALA [31] or with the HKL2000 suite

c The Authors Journal compilation 
c 2014 Biochemical Society
of programs [32]. Chain A of the original Mtb-AnPRT–PRPP
complex structure (PDB code 1ZVW) [22], without ligands, was
used as the search model for structure determination by molecular
replacement using MOLREP [33], and as the initial model
for refinement of the AnPRT–anthranilate analogue complex
structures. Refinement and model building was performed with
Coot [34] and Refmac5 [35], or autoBUSTER [36], with restraints
for the anthranilate analogues generated by using the PRODRG
server [37]. Restraints on bond lengths and angles were based
on the ideal values of Engh and Huber [38] and model quality
was assessed using MolProbity [39]. Full data collection and
refinement statistics are given in Supplementary Table S1.
Figures illustrating structural details were prepared using PyMOL
(Schrödinger; http://www.pymol.org), with maps generated using
the FFT program [40] in the CCP4 suite [41].
The crystallographic co-ordinates and structure factors have
been deposited in the RCSB PDB (Supplementary Table S1).
RESULTS
Mtb -AnPRT catalyses a sequential reaction and is inhibited by high
levels of its substrate anthranilate
To examine the kinetic mechanisms of the reaction, initial rate
measurements were taken at various concentrations of both
substrates. The kinetic profile is entirely consistent with a
sequential mechanism for this enzyme (Figure 2A). In addition,
for assays performed at higher concentrations of the substrate
anthranilate, a significant fall in the enzyme activity was observed,
indicating that the enzyme is subject to significant substrate
inhibition (Figure 2B and Table 1).
Fluorine- and methyl-substituted analogues of anthranilate act as
alternative substrates for Mtb -AnPRT
In the process of screening a targeted library of 165 compounds
for inhibitors of Mtb-AnPRT, we discovered several compounds,
including 5MA (5-methylanthranilate) and orthanilate, that are
alternative substrates for the enzyme [23]. The fluoroanthranilates
inhibit mycobacterial growth in a tryptophan-dependent manner
[5] and we established that 4FA (4-fluoroanthranilate) was also
an alternative substrate of Mtb-AnPRT. These results prompted
us to investigate a series of anthranilate analogues, with fluorine-
or methyl-substitutions on the aromatic ring, as substrates
for Mtb-AnPRT (Table 1, and Supplementary Figures S1 and
S2 at http://www.biochemj.org/bj/461/bj4610087add.htm). The
anthranilate analogue with C6 fluorine substitution has been
identified previously as inhibiting Pseudomonas aeruginosa
pathogenesis in mice [42], and both C5 and C6 fluoroanthranilate
analogues have shown promising bactericidal activity against
M. tuberculosis in the absence of tryptophan [5]. Orthanilate,
which carries a sulfonyl group in place of the carboxy group of
anthranilate, was also tested in the present study. The ability of
Mtb-AnPRT to process these alternative substrates was probed
using a combination of UV-based assays, MS and 1 H-NMR
spectroscopy.
In order to assess the ability of Mtb-AnPRT to process
each anthranilate analogue, a coupled UV-based assay for
PPi production was used. PPi production by Mtb-AnPRT was
observed in the presence of all alternative substrates and PRPP,
and kinetic parameters were determined for these anthranilate
analogues (Table 1).
Fluorine substitution around the aromatic ring of anthranilate
had a relatively minor effect on the rate of catalysis, with a
 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase
Figure 2 Plots for determination of the catalytic mechanism and
anthranilate kinetic parameters
(A) Mtb -AnPRT kinetic data obtained using the enzyme-coupled UV assay, fitted to the ternary
mechanism equation and presented as a double-reciprocal plot. PRPP was used at 12.5 μM
(䊐), 25 μM () or 62.5 μM (䊊). The K m values for anthranilate and PRPP were determined to
be 0.9 + + + −1
− 0.6 μM and 12 − 4 μM respectively, with a k cat value of 1.1 − 0.1 s . (B) Substrate
inhibition is observed at high concentrations of anthranilate, with a K i value of 45 +
− 6 μM.
Results were obtained using the pyrophosphate UV assay in the presence of 120 μM PRPP,
and fitted to the substrate inhibition equation. (C) Michaelis–Menten plot for determination
of the apparent K m of anthranilate, as measured by the pyrophosphate assay in the presence
of 120 μM PRPP. At lower concentrations of anthranilate, no inhibition is observed, with an
apparent K m value for anthranilate of 1.7 + + −1
− 0.1 μM, and a k cat value of 2.0 − 0.1 s .
91
decrease in kcat values of 25–40 %. The apparent K m for the
fluorinated analogues, however, is highly dependent on the site
of substitution, with fluorine substitution on C3 or C5 resulting
in an 18- and 6-fold increase in K m respectively, whereas fluorine
substitution on either C4 or C6 produces similar K m values to
that for anthranilate. The biggest decrease in kcat /K m is observed
for the alternative substrate 3FA, where the electron-withdrawing
fluorine substituent is found adjacent to the nucleophilic amino
group. K i values for substrate inhibition by these analogues are
again highly dependent on the site of substitution, but show a
different trend, with fluorine substitution on C3 and C4 resulting
in a 24- and 8-fold increase in K i respectively compared with
anthranilate, but substitution on either C5 or C6 having relatively
little effect. Of the anthranilate analogues tested in the present
study, 6FA has kinetic parameters that are most similar to those
of anthranilate.
Methylation of the anthranilate ring has a more significant
effect on the rate of catalysis, with this substitution at C3, C5
or C6 resulting in a ∼3-fold reduction in kcat , and substitution at
C4 yielding a 10-fold reduction. All of the methylated analogues
have much poorer affinity for the enzyme, with apparent K m values
that are increased by at least two orders of magnitude relative
to that of anthranilate. Substrate inhibition was not observed
with these analogues, although this analysis was limited by the
concentrations of methylated analogue required.
1
H-NMR spectroscopy confirms the production of substituted PRA
products from anthranilate analogues
To confirm that the hydrolysis of PPi in the presence
of the anthranilate analogues and PRPP indeed results in
product formation, the enzymatic production of substituted
PRA analogues by Mtb-AnPRT was followed using 1 H-NMR
spectroscopy. Anthranilate, and its substituted analogues, were
each incubated with an excess of PRPP and Mtb-AnPRT, and
1
H-NMR spectra were recorded at 100–300 s intervals as the
reactions proceeded. In the reaction with the native substrate
anthranilate, there was a clear consumption of PRPP over time,
as demonstrated by the loss of signal at 5.80 p.p.m. due to its
anomeric proton, and a corresponding gain of signals between 6.8
and 7.8 p.p.m. and between 5.3 and 5.7 p.p.m., which are due to
PRA (Figure 3, and Supplementary Figure S3 and Supplementary
Table S2 at http://www.biochemj.org/bj/461/bj4610087add.htm).
As observed previously [43], PRA is unstable and was observed to
break down to anthranilate and ribose, with a peak concentration
of PRA observed after approximately 12 min. The anthranilate
released again forms a substrate for Mtb-AnPRT, allowing the
PRA formation reaction to proceed until all of the PRPP is
consumed (Figure 3A).
Unexpectedly, two anomeric protons (at 5.70 and 5.31 p.p.m.)
and two sets of aromatic proton peaks were identified
corresponding to the product PRA. This indicates that both
α- and β-epimers at the C1 position of the ribose moiety
of PRA are present in the product mixture. The 5.70 and
5.31 p.p.m. resonances were assigned to the α- and β-epimers
of PRA respectively on the basis of chemical shifts and coupling
constants reported for related compounds [44,45]. From the
relative integrals of the two anomeric proton peaks, the ratio of
the α- to the β-epimer is approximately 7:3 at equilibrium under
the conditions of the assay.
As with other PRT enzymes [46,47], it is reasonable to
expect that the Mtb-AnPRT reaction proceeds with inversion of
stereochemistry at the C1 position on the ribose ring of PRPP.
Crystal structures of several different AnPRT enzymes [19,22,25]

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92 T. V. M. Cookson and others

Table 1 Apparent K m values, turnover number, specificity constant and substrate inhibition K i values for substituted anthranilate substrates of Mtb -AnPRT
N/A indicates that the value was not measured, as no substrate inhibition was observable under assay conditions. The Michaelis–Menten plots are shown in Supplementary Figures S1 and S2 at
http://www.biochemj.org/bj/461/bj4610087add.htm.

Substrate Structure K m value (μM) k cat value (s − 1 ) k cat /K m (s − 1 · μM − 1 ) K i value (μM)

Anthranilate 1.7 +
− 0.1 2.0 +
− 0.1 1.2 +
− 0.1 45 +
−6

3FA 31 +
−3 1.4 +
− 0.03 0.05 +
− 0.006 1090 +
− 330

4FA 2.1 +
− 0.2 1.2 +
− 0.1 0.6 +
− 0.1 380 +
− 100

5FA 10 +
−1 1.3 +
− 0.1 0.13 +
− 0.01 60 +
− 11

6FA 1.4 +
− 0.1 1.3 +
− 0.1 0.9 +
− 0.1 34 +
−5

3MA 290 +
− 20 0.63 +
− 0.03 0.002 +
− 0.0002 N/A

4MA 490 +
− 130 0.15 +
− 0.02 0.0003 +
− 0.0001 N/A

5MA 570 +
− 120 0.62 +
− 0.07 0.0011 +
− 0.0004 N/A

6MA 800 +
− 120 0.71 +
− 0.07 0.0009 +
− 0.0002 N/A

Orthanilate 130 +
− 10 0.1 +
− 0.004 0.0008 +
− 0.0001 N/A

provide supporting evidence for this claim, as it can be seen that
anthranilate can only approach from the β-position, thereby only
creating β-PRA. It is therefore likely that the α-PRA observed
is formed after the reaction due to rapid epimerization of β-
PRA in solution, a phenomenon that has been identified in other
PRA-like compounds such as phosphoribosyl amine [44,48].
Epimerization is likely to occur via intramolecular rearrange-
ment through an imine intermediate (Supplementary Figure
S4 at http://www.biochemj.org/bj/461/bj4610087add.htm). The
products of the Mtb-AnPRT reaction, formed in the presence of
NaCNBH3 to trap the proposed iminium ion intermediate, were
analysed using MS. Evidence for the reduced imine was indeed
observed, with m/z peaks present at 352.0782 and 374.0603 Da,
corresponding to the masses of the reduced imine and its sodium
salt respectively.
The Mtb-AnPRT-catalysed turnover of PRPP in the presence
of all of the anthranilate analogues was followed using 1 H-NMR
spectroscopy. For all of these alternative substrates, excluding
3MA, the concurrent formation of the corresponding PRA ana-
logue was detected (Supplementary Figure S5 and Supplementary
Table S2 at http://www.biochemj.org/bj/461/bj4610087add.htm).
As observed for native PRA, the fluorine- and methyl-substituted


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 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase
Figure 3 Progress curves for Mtb -AnPRT-catalysed reactions followed by 1 H-NMR spectroscopy
PRPP (2.5 mM) and anthranilate (A) or 4FA (B) (1 mM) were incubated with Mtb -AnPRT (0.8 μM) at 25 ◦ C and pH 8, and 1 H-NMR spectra were collected at 105-s intervals. Approximate
concentrations during the reaction were estimated on the basis of peak integrals for H1 of PRPP, H8 of anthranilate and H8 of PRA (α-PRA and β-PRA combined), relative to an internal TSP
(trimethylsilyl propionate) standard. Full details of the 1 H-NMR assay are given in the Experimental section.
PRAs accumulate as a mixture of α- and β-epimers in an
approximate 7:3 ratio in the assay solution.
Fluorinated and sulfonylated analogues of PRA were observed
to be significantly more stable than PRA itself. As illustrated
for 4FA in Figure 3(B), the stability of the product, 4F-PRA
(4-fluoro-PRA), results in a more distinct biphasic progress
curve during monitoring of the Mtb-AnPRT-catalysed reaction
by NMR spectroscopy. Initially, the reaction progresses quickly
until 4FA is consumed, and then it continues at a lower
rate governed by the breakdown of 4F-PRA to release 4FA,
until the excess PRPP is consumed. Conversely, the inability
to detect the PRA analogue resulting from turnover of 3MA
may reflect a shorter half-life of 3M-PRA (3-methyl-PRA)
relative to PRA itself. NMR spectroscopy was also used to
monitor directly the degradation of PRA and two substituted
PRA analogues in solution (Supplementary Figure S6 at
http://www.biochemj.org/bj/461/bj4610087add.htm).
The substituted PRA analogues were also shown to be
substrates for the next two enzymes of the tryptophan biosynthesis
pathway from E. coli PRAI and InGPS, which are expressed
as a single protein with two catalytic domains [49]. The
formation of fluorinated and methylated InGP analogues was
observed by MS, UV spectroscopy and 1 H-NMR spectroscopy
(Supplementary Figures S7–S9 and Supplementary Table S3
at http://www.biochemj.org/bj/461/bj4610087add.htm) in the
presence of Eco-PRAI–InGPS. In the 1 H-NMR experiments,
it was observed that both epimers of PRA or 4F-PRA were
consumed in line with the predicted rapid interconversion
of these isomers. Unlike the other PRA analogues studied,
sulfonyl-PRA could theoretically undergo PRA isomerization
catalysed by PRAI to yield CdRP [1-(o-carboxyphenylamino)-
1 -deoxyribulose-5 -phosphate], but not the decarboxylation
catalysed by InGPS to form the indole InGP. Consistent with
this, the formation of InGP was not observed on incubation of
sulfonyl-PRA with Eco-PRAI–InGPS. However, the formation
of a small amount of an uncharacterized compound or
compounds was indicated by the appearance of a new set
of 1 H-NMR peaks (Supplementary Figure S8 at http://www.
biochemj.org/bj/461/bj4610087add.htm).
93
Structures of Mtb -AnPRT in complex with fluorine- and
methyl-substituted anthranilates define two distinct
substrate-binding sites
Structures of Mtb-AnPRT soaked with the anthranilate analogues
for various time periods prior to crystal harvesting and freezing
were determined to gain structural insight into the binding of these
alternative substrates. The lower turnover numbers of the enzyme
with these analogues has allowed substrate-bound complexes to be
visualized, which are difficult to access with the natural substrate
anthranilate. The structures of these complexes reveal a consistent
pattern where S3, the substrate-capture site, is occupied first, after
which the substrate moves through the tunnel to a position adja-
cent to PRPP, the catalytic site (S1). The existence of more than
one binding site for anthranilate has previously been inferred from
modelling [22] and inhibition [23,26] studies and by analogy with
results for the Sso-AnPRT enzyme [25]. Notably, however, no dir-
ect structural information for Mtb-AnPRT is yet available showing
the substrate anthranilate bound in a catalytically relevant mode.
The structure of Mtb-AnPRT soaked with PRPP, Mg2 + and
4FA for 6 min most clearly defines the two major sites S1 and S3
(Figure 4), and is used here as the reference structure to describe
the active site and understand the reaction mechanism. In this
complex, both sites are fully occupied by the 4FA substrate and
the same interactions are made between enzyme and substrate in
both molecules of the Mtb-AnPRT dimer. The enzyme structure
is unchanged from its ligand-free state by substrate binding, and
no domain movements are necessary to bring the anthranilate
substrate into contact with the PRPP as was proposed for Sso-
AnPRT [25]. Superposition of the 4FA complex on to the
substrate-free and liganded (with PRPP, Mg2 + and inhibitors)
Mtb-AnPRT structures previously described [23] gave RMSDs of
0.22–0.88 Å for all Cα atoms.
The pocket within which the substrate binds in its catalytically
competent S1 position is bounded by the PRPP ribose ring and
residues from the β1–α5 and β2–α6 loops that close over PRPP
(Val106 , Gly107 , Thr108 , His136 and Asn138 ), together with Ala179 ,
Arg193 , Gly206 and Pro207 . The binding mode of the 4FA molecule
at S1 is shown in Figure 4(C). It has several interactions with

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94 T. V. M. Cookson and others

Figure 4 Structure of Mtb -AnPRT soaked with PRPP, Mg2 + and 4FA for 6 min
4FA is shown bound in both the inner catalytic S1 (orange) and the outer S3 (yellow) of chain A. PRPP is shown in magenta, with Mg2 + as black spheres. Polar contacts are shown as yellow
broken lines, and water molecules as red spheres. Analogous binding modes and interactions were observed for chain B. (A) Monomer of Mtb -AnPRT with PRPP, Mg2 + and 4FA bound, and with
key structural features indicated. (B) Surface representation showing 4FA bound at S1 and S3. (C) 4FA bound in the inner catalytic site (S1). (D) 4FA bound in the outer site (S3). (E) Omit map
calculated before the addition of ligands to the model, where the F o − F c map is contoured at 3σ and coloured green and red for positive and negative density respectively. (F) The 2F o − F c map
for the ligands after final refinement is shown in blue, contoured at 1σ .


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 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase
the surrounding structure including hydrogen bonds between its
amino group and the carbonyl oxygen of Gly107 and the C2
hydroxy group of PRPP, and hydrogen bonds made between
its carboxy group, the side-chain amide group of Asn138 , the η-
nitrogen of Arg193 and several water molecules. The other notable
interaction involves the aromatic ring, which is sandwiched
between the Cα of Gly206 and the Cβ of Asn138 . In this orientation,
the amino nitrogen is 4.3 Å from the C1 atom of the PRPP
ribose ring, in position to attack this atom from the β position.
The 4-fluoro-substituent of 4FA is well accommodated, making
hydrophobic contacts with Val106 Cγ 2 (3.2 Å) and with the side
chain of His136 (3.2–3.4 Å). The same binding mode is seen for the
5FA structure, where the fluorine moiety is situated ∼3.5 Å from
the centre of the His136 imidazole ring. Structures of Mtb-AnPRT
with 6FA or 6MA bound also show binding at S1, where the C6-
substituent projects unimpeded along the substrate tunnel. The
accommodation of 4FA, 5FA and 6FA at S1 is consistent with the
similarity of their kinetic parameters to that of the native substrate,
anthranilate. Crystal structures of complexes with 3MA, 4MA and
5MA did not show binding at S1, and may be indicative of steric
clashes amplified by the larger methyl substitutions. Likewise,
3FA accommodated in this site would result in unfavourable
electrostatic interactions between the fluorine substituent and the
Gly107 backbone carbonyl.
The second binding site of 4FA, which we designate the
substrate-capture site, is located ∼8 Å from S1 (distance measured
between the centres of the two rings), in a position at the
entrance to the tunnel that extends into the active site. This site is
different from a more buried site (S2) that was predicted by Mtb-
AnPRT modelling [22] and which is occupied by anthranilate
in Sso-AnPRT structures [25], but is coincident with the outer
inhibitor-binding site (S3) seen for many inhibitor complexes of
Mtb-AnPRT [23,26]. That it differs in detail from the second
anthranilate site found for Sso-AnPRT [25] is not surprising
given that the residues involved are mostly non-conserved. In S3
(Figure 4D), the substrate is held in position by stacking between
Pro180 on one side and the side chains of Tyr186 and Ala190 on the
other, and by hydrogen bonds between its carboxy group, Arg194
and two waters, and between its amino group, a water molecule
and the carbonyl oxygen of His183 .
Early binding events as deduced from the substrate complex
structures
All the crystal structures of Mtb-AnPRT complexed with the
fluorine- and methyl-substituted anthranilates show binding at
S3, even for the very shortest soaking times. This clearly points
to binding at this site as the earliest event in substrate binding. We
illustrate this for the 4FA substrate after a 6 s soak (Figure 5). For
this complex, the binding mode in S3 is already well defined for
both Mtb-AnPRT molecules of the dimer, with the substrate in the
same orientation as seen in the longer 6-min 4FA-soaked complex.
When the crystal structures of the other substrate complexes are
compared, the substrate is always bound in S3 with its anthranilate
ring stacked between Pro180 and Tyr186 as for the 4FA complex, and
with its carboxy group hydrogen-bonded to Arg194 . The substrate’s
aromatic ring can, however, be flipped to give two different modes
of interaction by the amino group, hydrogen-bonded either to the
backbone oxygen of His183 (4FA, 5FA, 4MA, 5MA and 6MA)
or to Oδ1 of Asn138 (3FA, 6FA and 3MA). It is possible that the
fluorine or methyl substituents force the substrate to adopt one of
two orientations in S3 to minimize unfavourable interactions with
the surrounding residues. These interactions and orientations of
the substrates at S3 could contribute to the variation in kinetic
95
parameters seen between substrates with substituent groups at
different positions.
The movement of anthranilate-like substrates from S3 into the
catalytic S1 is more complex than it appears at first sight. At
S3, the amino group for a number of the alternative substrates
is oriented towards His183 , hydrogen-bonding to the backbone
carbonyl oxygen. The ring must be flipped 180◦ , however, to
orient the amino group towards the PRPP molecule in the substrate
pocket. This could happen by normal ‘breathing’ of the protein
structure. However, the structure of the 6MA complex after
a 30 min soak shows positive electron density at a third site,
equivalent to the previously described S2 [26], which is located
between S1 and S3 (Figure 5E). S2 is formed in a mostly
hydrophobic pocket between the side chains of Met86 , Ala179 ,
His183 , Tyr186 and His136 . The electron density at this site is more
poorly defined, and cannot be accurately modelled, suggesting
that it is a transient site that lacks anchor points for the substrate
molecule.
Mtb-AnPRT soaked with PRPP, Mg2 + and anthranilate for
4 s shows electron density present in the substrate-capture site
alone. The observation that anthranilate can bind in this outer
site without PRPP or Mg2 + present provides some insight into
the order of substrate-binding events, and is consistent with
the recent report by Evans et al. [26] that a bi-anthranilate-like
inhibitor can bind in both S2 and S3 in the absence of PRPP
or Mg2 + . Previous comparisons of the Mtb-AnPRT PRPP-bound
and ligand-free structures [22] show clear rearrangement of the
flexible β1–α5 and β2–α6 loops upon PRPP/Mg2 + binding, and
these rearrangements are likely to be necessary to construct the
inner catalytic binding site for anthranilate. These flexible loops
remain disordered in the 4-s anthranilate soak structure, indicating
that binding to the outer site S3 does not require prior PRPP/Mg2 +
binding or lead to the rearrangement of the loops. Binding of
anthranilate at S3 may precede PRPP/Mg2 + binding, with the
enzyme effectively sequestering this substrate until it can proceed
to the inner catalytic site S1 to enable reaction with PRPP.
DISCUSSION
PRTs are present in the biosynthetic pathways of tryptophan
and histidine, as well as playing a role in nucleotide-salvage
and -synthesis pathways, and as such are important enzymes
for bacterial metabolism. Their potential as antibacterial drug
targets makes the understanding of their mechanisms of significant
interest. The present study provides insight into the reaction
mechanism of Mtb-AnPRT, and includes the first crystal structures
of Mtb-AnPRT in complex with both PRPP and anthranilate
analogues. These structures provide a clearer picture of how
anthranilate is transported from the bulk solvent to the catalytic
pocket for a productive reaction with PRPP.
Mtb-AnPRT catalyses a sequential reaction and catalytic
activity is inhibited by high concentrations of the substrate
anthranilate. Mtb-AnPRT is remarkably tolerant of substitution on
the phenyl ring of anthranilate. Fluorine- and methyl-substituted
analogues of anthranilate were accepted as alternative substrates,
and substituted PRA products were identified by NMR and
MS studies. Unsurprisingly, with its lower steric demands,
fluorine substitution was more efficiently accommodated than the
introduction of a methyl group. The bactericidal activity of 5FA
and 6FA against M. tuberculosis demonstrated by Zhang et al. [5]
is only observed in the absence of tryptophan, consistent with
these compounds acting by targeting tryptophan biosynthesis.
This activity could plausibly originate from the toxicity of
fluorinated intermediates generated by the turnover of these
compounds by Mtb-AnPRT and subsequent pathway enzymes.

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96 T. V. M. Cookson and others

Figure 5 Structures of Mtb -AnPRT soaked with PRPP, Mg2 + and either 6MA for 30 min or 4FA for 6 s
6MA is shown bound at both S1 (cyan) and S3 (purple) of chain A, and 4FA (green) is shown bound at S3 of chain B. PRPP is shown in yellow, with Mg2 + as green spheres. Polar contacts are
shown as yellow broken lines, and water molecules as red spheres. Analogous binding modes and interactions were observed for chain B of the 6MA structure, and in chain A of the 4FA structure. (A
and B) Binding of ligands in the active site for the 6MA and 4FA structures respectively. (C and D) 2F o − F c maps for the ligands after final refinement for the 6MA and 4FA structures respectively.
The 2F o − F c maps are shown in blue and are contoured at 1σ . (E and F) Omit maps for the 6MA and 4FA structures respectively. The omit maps displayed were calculated before the addition of
ligands to the models, and show the F o − F c map contoured at 3σ and coloured green and red for positive and negative density respectively.

In crystal structures of Mtb-AnPRT reported previously [22,23],
it has been observed that residues from the flexible loops β1–
α5 and β2–α6 that close over the PRPP molecule after binding
into the substrate pocket form the contacts for anthranilate
binding in S1. This observation, coupled with our findings of
a sequential mechanism, strongly indicates that anthranilate can
only productively bind at the inner site S1 after PRPP has bound.
The crystal structures solved in the present study strengthen
this claim further, as 4FA can clearly be seen to bind to the
residues from the flexible loops at S1, namely Asn138 and Gly107 .
It can also be observed that the above-mentioned flexible PRPP-
binding loops are in the ‘open’ position in the ligand-free structure


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 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase
and closed in the 4FA/PRPP/Mg2 + -bound and PRPP/Mg2 + -
bound structures, indicating that the enzyme is only primed for
efficient anthranilate binding at the catalytic S1 once PRPP is
bound. In contrast, the AnPRT enzymes from S. solfataricus
and Saccharomyces cerevisiae have both been reported to exhibit
random sequential Bi Bi mechanisms [25,50]. For Sso-AnPRT,
this discrepancy is surprising given the similarity of the substrate-
binding modes between the two structures.
The closure of flexible loops over a substrate-binding pocket
in Mtb-AnPRT is not unique among the PRT family. Substrate
binding instigates conformational change in both type I and
type II PRTs, either by flexible loop rearrangement (type I) or by
relative domain rotation (type II). In both cases, the importance
of enclosing the active site has been correlated with protecting the
hypothesized highly reactive carbocation intermediate. Previous
experiments on orotate PRT, ATP-PRT and hypoxanthine PRT
using KIEs strongly support the idea of an SN 1-like reaction
[12,13]. The observation that none of the fluoroanthranilate
analogues in the present study significantly adversely affects
the overall kcat value of Mtb-AnPRT could indicate that the
nucleophilicity of the amino group is not of great significance
to the overall reaction rate, consistent with either a dissociative
SN 1-like mechanism for AnPRT or rate-limiting substrate-binding
or release steps.
The substituted analogues allow direct observation of two
distinct binding sites for anthranilate in Mtb-AnPRT: an inner
catalytic site (S1) and an outer substrate-capture site (S3).
Additionally, some evidence for passage of anthranilate between
the outer S3 and the catalytic S1 is seen via an intermediate
binding site (S2), in a manner consistent with previously observed
inhibitor-binding studies [26]. The role of S3 in trapping
anthranilate and the flexibility of the loops associated with the
active site may have a direct role in the productive use of
highly reactive PRPP in the transferase reaction. If anthranilate
is first captured in the outer S3 and PRPP then binds, leading
to concomitant formation of the higher-affinity inner catalytic
site by loop rearrangements, efficient shuttling of anthranilate
to the catalytically relevant S1 is achieved. This sequence of
events allows anthranilate to efficiently react with the developing
oxocarbenium ion intermediate derived from PRPP and
minimizes exposure of this highly reactive intermediate to water.
Although potentially facilitating efficient formation of PRA
by AnPRT, the outer S3 may also be somewhat of an Achilles
heel for the enzyme. These studies reveal that the substrate
anthranilate significantly inhibits this enzyme, and it is tempting
to speculate that this inhibition may arise from non-productive
binding of anthranilate to S3 as anthranilate is transferred to the
productive catalytic S1. It is noteworthy that bactericidal 5FA and
6FA differ from the other alternative substrates in their ability to
mimic the anthranilate substrate inhibition, and this contrasting
kinetic behaviour is suggestive of different affinities of the two
anthranilate sites for these analogues. Moreover, several inhibitors
of this enzyme have been shown to utilize S2 and S3 [23,26],
suggesting that this unique allosteric feature of this enzyme may
be fruitfully targeted for inhibition, and may be the basis for new
anti-TB therapeutics.
AUTHOR CONTRIBUTION
Shaun Lott, Tammie Cookson, Alina Castell, Esther Bulloch and Emily Parker conceived and
designed the experiments. Tammie Cookson, Alina Castell, Esther Bulloch and Francesca
Short performed the experiments. Tammie Cookson, Alina Castell, Esther Bulloch and
Emily Parker analysed the data. Emily Parker, Tammie Cookson, Alina Castell, Esther
Bulloch, Genevieve Evans and Edward Baker wrote the paper. Alina Castell, Genevieve
97
Evans, Tammie Cookson, Esther Bulloch, Emily Parker, Edward Baker and Shaun Lott
revised the paper before acceptance.
ACKNOWLEDGEMENTS
We gratefully acknowledge Dr Joseph Mire, Dr Inna Kriger and Professor Jim Sacchettini
(Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX,
U.S.A.) for the phenotypic high-throughput screens that identified 4FA, initial tryptophan
biosynthesis inhibition experiments involving 4FA and for providing us with a small
amount of 4FA compound for initial experiments. The Mtb -AnPRT expression plasmid was
originally supplied by Dr Farah Javid-Majd (Department of Biochemistry and Biophysics,
Texas A&M University). We also acknowledge Dr Jackie Kendall, Dr Julie Spicer and
Professor Bill Denny (Auckland Cancer Society Research Centre, Auckland, New Zealand)
for providing the compounds for the initial targeted screen against Mtb -AnPRT, and for the
5MA, 6MA and orthanilate compounds tested in the present study. This work was performed
as part of the M. tuberculosis Structural Genomics Consortium (http://www.webtb.org).
FUNDING
The research was funded by grants from the Health Research Council of New Zealand,
the Foundation for Research, Science and Technology of New Zealand and the Tertiary
Education Commission of New Zealand, through the Maurice Wilkins Centre.
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Biochem. J. (2014) 461, 87–98 (Printed in Great Britain) doi:10.1042/BJ20140209

SUPPLEMENTARY ONLINE DATA

Alternative substrates reveal catalytic cycle and key binding events in the
reaction catalysed by anthranilate phosphoribosyltransferase from
Mycobacterium tuberculosis
Tammie V. M. COOKSON*1 , Alina CASTELL†1, 2 , Esther M. M. BULLOCH†, Genevieve L. EVANS†, Francesca L. SHORT†3 ,
Edward N. BAKER†, J. Shaun LOTT†4 and Emily J. PARKER*4
*Maurice Wilkins Centre for Molecular Biodiscovery, Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8140,
New Zealand
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand

Figures S1–S9 and Tables S1–S3 appear on the following pages.

1
These authors contributed equally to this work.
2
Present address: Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology, 171 77 Stockholm, Sweden.
3
Present address: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
4
Correspondence may be addressed to either of these authors (email s.lott@auckland.ac.nz or emily.parker@canterbury.ac.nz).
The structural co-ordinates reported for Mycobacterium tuberculosis anthranilate phosphoribosyltransferase complexed with anthranilate or anthranilate
analogues have been deposited in the PDB under codes 4N5V, 4N8Q, 4N93, 4OWM, 4OWN, 4OWO, 4OWQ, 4OWS, 4OWU and 4OWV.


c The Authors Journal compilation 
c 2014 Biochemical Society
 T. V. M. Cookson and others

Figure S1 Michaelis–Menten graphs for calculation of K m values for alternative substrates of Mtb -AnPRT
Results were obtained using the EnzChek® Pyrophosphate Assay Kit with PRPP held at a saturating concentration of 120 μM. Reactions were initiated with the addition of anthranilate/alternative
substrate. Results were plotted using GraFit 5.


c The Authors Journal compilation 
c 2014 Biochemical Society
 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase

Figure S2 Substrate inhibition graphs for calculation of K i values for alternative substrates of Mtb -AnPRT
Results were obtained using the EnzChek® Pyrophosphate Assay Kit with PRPP held at a saturating concentration of 120 μM. Reactions were initiated with the addition of anthranilate/alternative
substrate. Results were plotted using GraFit 5.


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 T. V. M. Cookson and others

Figure S3 Monitoring reaction catalysed by Mtb -AnPRT using 1 H-NMR spectroscopy

(A) Spectrum of assay solution containing α-PRPP (2.5 mM) and anthranilate (1 mM) at pH 8 before incubation with Mtb -AnPRT. Note that the commercially obtained α-PRPP also contains β-PRPP
as an impurity, which is not a substrate for Mtb -AnPRT. (B) Spectrum of assay solution following incubation with Mtb -AnPRT (0.8 μM) at 25 ◦ C for 10 min. The substrates PRPP and anthranilate
have been consumed, and both α-PRA and β-PRA are present at an approximate 7:3 molar ratio. Chemical shifts are given relative to TSP (δ 0 p.p.m.). Full details of the NMR assay composition
and collection of 1 H-NMR spectra are given in the Experimental section of the main text.

Figure S4 Epimerization of the Mtb -AnPRT product PRA via an imine intermediate and its reduction to the open-chain form


c The Authors Journal compilation 
c 2014 Biochemical Society
 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase

1
Figure S5 H NMR spectra for the production of substituted PRAs from anthranilate analogues
The upper spectrum of each panel shows the anthranilate analogue (1 mM) and PRPP (2 mM) before the addition of Mtb -AnPRT. The lower 1 H-NMR spectrum is following incubation of this sample
with 0.2 μM Mtb -AnPRT for 20 min at 25 ◦ C. Full details of the NMR assays are given in the Experimental section of the main text.

c The Authors Journal compilation 
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 T. V. M. Cookson and others

Figure S6 Decomposition of PRA and PRA analogues as followed by 1 H-

NMR spectroscopy
PRPP (2.5 mM) and anthranilate or analogue (1 mM) was incubated with 0.8 μM Mtb -AnPRT to
form PRA or PRA analogue. Following consumption of the substrates, the rate of decomposition
of PRA or PRA analogue was monitored at 25 ◦ C and at pH 8 by collecting 1 H-NMR spectra at
1.5–5 min intervals. Rates of degradation were measured on the basis of peak integrals for H8 of
PRA (α-PRA and β-PRA combined), or analogue, relative to an internal TSP standard. Results
were fitted to a first-order exponential decay curve to determine the half-life of each compound.
It has been noted previously that PRA undergoes hydrolysis to yield ribose 5-phosphate and
anthranilate in a first-order exponential decay process with a half-life of 450 s at 25 ◦ C and
pH 7.5 [3]. By following the rate of decrease in the 1 H-NMR resonance of H8 of PRA, after
the consumption of all the PRPP substrate, a half-life of 580 + − 10 s for PRA was determined
under the conditions of our 1 H-NMR spectroscopy assay at 25 ◦ C and pH 8. This analysis was
repeated for 4F-PRA and sulfonyl-PRA, which are formed from the action of Mtb -AnPRT on 4FA
and orthanilate respectively. Both of these analogues are substantially stabilized relative to native
PRA, with half-lives of 2960 + +
− 50 and 6610 − 30 s determined for 4F-PRA and sulfonyl-PRA
respectively.

Figure S7 Wavelength scans of the Mtb -AnPRT/Eco -PRAI–InGPS reaction

for fluoro-/methyl-anthranilate with PRPP
The absorbance can be seen to increase in the range 260–290 nm for each substrate. Solutions
for analysis of FA contained 1 mM MgCl2 , 0.1 μM Mtb -AnPRT, 1.7 μM Eco -PRAI–InGPS,
0.6 mM PRPP and 100 μM FA (3FA, 4FA, 5FA or 6FA). Solutions for analysis of MA contained
1 mM MgCl2 , 30 μM Mtb -AnPRT, 150 μM Eco -PRAI–InGPS, 0.6 mM PRPP and 0.5 mM MA
(5MA or 6MA). Reactions were scanned from 360 to 220 nm with a cycle length time of 2.5 min.
Solutions were thermally equilibrated for 5 min before initiation with alternative substrate and
either allowed to go to completion or to a maximum of 50 cycles.


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 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase

Figure S8 Turnover of PRA analogues by Eco-PRAI–InGPS monitored by 1 H-NMR spectroscopy

(A) 4F-PRA was formed by the incubation of 4FA (1 mM) and PRPP (2.5 mM) with Mtb -AnPRT (0.8 μM). (B) Addition of Eco -PRAI–InGPS (2.5 μM) to this 4FA-PRA sample resulted in
complete conversion into 4-fluoro-InGP. δ 7.76 (dd, 5.5, 8.8 Hz, H6), 7.42 (s, H1 ), 7.25 (dd, 10.1, 2.2 Hz, H3), 6.98 (ddd, 2.2, 9.4 Hz, H5), 5.06 (d, 8.2 Hz, H3 ). (C) Sulfonyl-PRA was
formed by the incubation of orthanilate (0.5 mM) and PRPP (2.5 mM) with Mtb -AnPRT (0.8 μM). (D) Addition of Eco -PRAI–InGPS (2.5 μM) to this sulfonyl-PRA sample resulted in the
production of a new set of minor resonances as indicates by the broken arrows (δ 7.70, 7.42, 6.84 and 6.74 p.p.m.). We speculate that these new resonances may correspond to the CdRP
[1-(o -carboxyphenylamino)-1 -deoxyribulose-5 -phosphate] analogue 1-(o -sulfonylphenylamino)-1-deoxyribulose-5-phosphate, formed from isomerization of sulfonyl-PRA by PRAI. Samples were
incubated at 25 ◦ C for approximately 10 min following the addition of each enzyme. Peaks marked by an asterisk result from an imidazole impurity present in the PRAI–InGPS preparation used for
these experiments. Full details of NMR assay composition and collection of 1 H-NMR spectra are given in the Experimental section of the main text.

1
Figure S9 H-NMR spectrum of InGP
PRA was formed by the incubation of anthranilate (1 mM) and PRPP (2.5 mM) with Mtb -AnPRT (0.8 μM). Addition of Eco -PRAI–InGPS (2.5 μM) and incubation at 25 ◦ C for approximately 10 min
resulted in complete conversion into InGP. Full details of NMR assay composition and collection of 1 H-NMR spectra are given in the Experimental section of the main text. δ 7.83 (d, 8 Hz, H6), 7.54
(d, 8.2 Hz, H3), 7.46 (s, H1 ), 7.27 (dd, 7.6 Hz, H4), 7.19 (dd, 7.5 Hz, H5), 5.10 (d, 8.2 Hz, H3 ).


c The Authors Journal compilation 
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c The Authors Journal compilation 

T. V. M. Cookson and others

Table S1 Data and refinement statistics for all complexes
Values for the outer resolution shell are shown in parentheses. R merge =  hklI (hkl ) − <I (hkl )>/ hklI (hkl ), where <I (hkl )> is the mean of the symmetry equivalent reflections of I (hkl ). The B factor is the average atomic temperature factor. R work =
|F o | − |F c |/|F o |. R free =  T |F o | − |F c |/ T |F o | (where T is a test dataset of 5 % of the total reflections randomly chosen and set aside before refinement). Ramachandran outliers were calculated using MolProbity [1]. Ideal RMSD values from Engh and Huber
[2]. NA, not applicable.
c 2014 Biochemical Society

4FA 4FA 6MA 3FA 5FA 6FA 3MA 4MA 5MA Anthranilate

Soak time 6 min 6s 30 min 2 min 5 min 5 min 2 min 2 min 4 min 4s
Data collection
PDB code 4N5V 4N8Q 4N93 4OWM 4OWN 4OWO 4OWQ 4OWS 4OWU 4OWV
Space group P 21 2 1 2 1 P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21
Cell dimensions
a , b , c (Å) 79.6, 92.5, 121.1 79.6, 91.0, 120.0 79.6, 92.2, 121.4 79.3, 92.0, 120.9 79.6, 92.3, 120.8 79.4, 92.1, 121.3 79.4, 92.3, 121.7 79.7, 91.8, 120.6 79.5, 91.8, 120.8 79.6, 91.7,
120.4
α, β, γ (◦ ) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90
Number of 71077 52754 58211 52873 52044 61270 71647 34257 71474 69457
unique
reflections
Resolution 73.52–1.90 120.0–2.08 121.4–2.03 120.9–1.99 120.8–2.11 121.3–1.99 121.7–1.89 120.6–2.43 120.8–1.89 120.4–1.90
range (Å) (1.95– 1.90) (2.20–2.08) (2.14–2.03) (2.10–1.99) (2.22–2.11) (2.10–1.99) (2.00–1.89) (2.56–2.43) (1.99–1.89) (2.00–1.90)
R merge 0.041 (0.483) 0.031 (0.203) 0.039 (0.233) 0.085 (0.485) 0.084 (0.496) 0.102 (0.494) 0.072 (0.480) 0.087 (0.495) 0.065 (0.485) 0.153 (1.047)
I /σ (I ) 14.7 (1.8) 17.2 (3.9) 17.2 (3.7) 12.2 (2.8) 17.9 (3.9) 14.0 (3.5) 17.8 (3.5) 17.6 (3.9) 20.6 (3.8) 8.2 (1.8)
Completeness 99.9 (100) 100 (99.9) 100 (100) 86.9 (90.0) 100 (100) 99.8 (99.4) 100 (100) 100 (100) 100 (100) 99.3 (99.7)
(%)
Multiplicity 2.0 (1.00) 2.0 (2.0) 2.0 (2.0) 4.6 (4.5) 7.2 (7.4) 7.3 (7.4) 7.3 (7.1) 7.2 (7.4) 7.2 (6.9) 5.0 (4.8)
Refinement
Number of
atoms, B
factor (Å2 )
Protein 5093, 25.7 5045, 33.4 5028, 22.7 5020, 21.8 4960, 25.7 5014, 19.8 4993, 21.9 4892, 33.4 4989, 21.4 4963, 19.9
Solvent 514, 35.1 294, 36.3 545, 32.9 453, 33.4 404, 35.8 513, 33.3 510, 34.5 204, 38.1 516, 35.1 485, 31.4
PRPP 44, 26.7 NA 44, 28.5 44, 35.4 22, 40.1 44, 27.0 44, 29.7 NA 44, 28.1 NA
Mg2 + 4, 29.3 NA 4, 27.2 4, 27.6 4, 28.2 4, 22.8 3, 26.4 NA 4, 22.9 NA
Analogue 44, 23.1 22, 47.0 44, 42.0 22, 43.1 44, 43.1 44, 27.2 22, 34.9 22, 34.7 22, 35.3 20, 46.4
Pyrophosphate NA NA NA NA 9, 27.8 NA NA 18, 64.0 NA NA
R work /R free 18.4/21.8 19.9/23.4 18.1/22.1 17.2/21.3 17.0/19.4 16.3/19.3 16.9/19.2 18.1/22.7 16.5/19.5 19.5/22.1
(%/%) (30.7/30.5) (23.3/28.7) (21.7/27.8) (24.2/28.1) (21.8/23.6) (22.5/26.4) (24.5/26.8) (23.8/32.4) (23.3/26.6) (40.6/43.7)
Ramachandran 0.29 0.59 0.29 0.29 0.29 0.29 0.29 0.30 0.29 0.29
outliers (%)
RMSD
Bond lengths 0.014 0.015 0.014 0.010 0.009 0.009 0.009 0.011 0.009 0.010
(Å)
Bond angles (◦ ) 1.417 1.476 1.462 1.454 1.441 1.441 1.479 1.469 1.475 1.436
 Alternative substrates of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase

Table S2 Chemical shift assignment of 1 H-NMR spectra of PRA and PRA analogues produced by Mtb -AnPRT
Chemical shifts are given relative to TSP (δ 0 p.p.m.). Full details of NMR assay composition and collection of 1 H NMR spectra are given in the Experimental section of the main text. ND, not detected;
NA, not applicable.

Assignment of chemical shifts (δ, p.p.m.) PRA 3M-PRA 4M-PRA 5M-PRA 6M-PRA 3F-PRA 4F-PRA 5F-PRA 6F-PRA Sulfonyl-PRA

H1 a 5.70 ND 5.67 5.66 5.60 5.71 5.62 5.65 5.62 5.69
H1 b 5.31 ND 5.29 5.28 5.23 5.39 5.24 5.25 5.25 5.32
H3a 7.05* ND 6.89 6.95* 6.85 NA 6.76* 7.02* 6.82 7.12*
H3b 6.88 6.84 6.81
H4a 7.41* ND NA 7.25* 7.16* 7.22* NA 7.17* 7.27* 7.47*
H4b
H5a 6.86* ND 6.70* NA 6.75 6.93* 6.55* NA 6.61* 6.93*
H5b 6.77
H6a 7.80* ND 7.71* 7.63 NA 7.55 7.83* 7.51 NA 7.73*
H6b 7.61 7.60
α-H1 a coupling constant (Hz) 3.7 ND 4.0 3.8 3.6 3.8 3.9 3.6 4.1 3.6
β-H1 coupling constant (Hz) 6.6 ND 6.4 6.4 6.9 5.4 6.7 6.8 6.9 6.7
Approximate ratio α/β 7:3 ND 6:4 7:3 7:3 7:3 7:3 7:3 7:3 8:2
*Peaks from the two epimers of PRA closely overlap preventing unique assignment. For PRA and the majority of PRA analogues, there was significant overlap in the aromatic peaks of the two
epimers, preventing unique assignment of these resonances to an epimer and complicating analysis of peak types, integrals and coupling constants in this region.

Table S3 Mass of substituted InGP products arising from turnover of anthranilate analogues by Mtb -AnPRT and Eco -PRAI–InGPS
Solutions contained 1.2 mM PRPP, 0.6 μM Mtb -AnPRT, 6 μM Eco -PRAI–InGPS, 2 mM MgCl2 and either 400 μM fluoro-anthranilate or 2 mM methyl-anthranilate. − indicates that the expected
product could not be observed.

Substrate Formula of product Expected mass of product (g/mol) Observed mass of product (g/mol)

Anthranilate C11 H14 NO6 PNa 310.0451 310.0441

6FA C11 H13 FNO6 PNa 328.0357 328.0348
5FA C11 H13 FNO6 PNa 328.0357 328.0349
4FA C11 H13 FNO6 PNa 328.0357 328.0347
3FA C11 H13 FNO6 PNa 328.0357 328.0350
6MA C12 H16 NO6 PNa 324.0607 324.0597
5MA C12 H16 NO6 PNa 324.0607 324.0600
4MA C12 H16 NO6 PNa 324.0607 324.0605
3MA C12 H16 NO6 PNa 324.0607 –

REFERENCES
1 Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J.,
Murray, L. W., Richardson, J. S. and Richardson, D. C. (2010) MolProbity: all-atom
structure validation for macromolecular crystallography. Acta Crystallogr. D Biol.
Crystallogr. 66, 12–21 CrossRef PubMed
2 Engh, R. A. and Huber, R. (1991) Accurate bond and angle parameters for X-ray
protein-structure refinement. Acta Crystallogr. A 47, 392–400 CrossRef
3 Sterner, R., Kleemann, G. R., Szadkowski, H., Lustig, A., Hennig, M. and Kirschner, K.
(1996) Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an
extremely stable and active homodimer. Protein Sci. 5, 2000–2008 CrossRef PubMed

Received 13 February 2014/7 April 2014; accepted 9 April 2014

Published as BJ Immediate Publication 9 April 2014, doi:10.1042/BJ20140209


c The Authors Journal compilation 
c 2014 Biochemical Society