Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis

Biochimica et Biophysica Acta 1794 (2009) 1510–1516

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Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis L.J. Domigan a, S.W. Scally b,c, M.J. Fogg d, C.A. Hutton b,e, M.A. Perugini b,c, R.C.J. Dobson a,b,c, A.C. Muscroft-Taylor a, J.A. Gerrard a,⁎, S.R.A. Devenish a,⁎ a

School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8020, New Zealand Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia Department of Biochemistry and Molecular Biology, University of Melbourne, VIC 3010, Australia d York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, UK e School of Chemistry, University of Melbourne, VIC 3010, Australia b c

a r t i c l e

i n f o

Article history: Received 27 April 2009 Received in revised form 5 June 2009 Accepted 25 June 2009 Available online 10 July 2009 Keywords: Dihydrodipicolinate synthase Lysine biosynthesis Bacillus anthracis Isothermal titration calorimetry Enzyme kinetics

a b s t r a c t Bacillus anthracis is a Gram-positive spore-forming bacterium that is the causative agent of anthrax disease. The use of anthrax as a bioweapon has increased pressure for the development of an effective treatment. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthetic pathway yielding two essential bacterial metabolites, meso-diaminopimelate (DAP) and (S)-lysine. DHDPS is therefore a potential antibiotic target, as microbes require either lysine or DAP as a component of the cell wall. This paper is the first biochemical description of DHDPS from B. anthracis. Enzyme kinetic analyses, isothermal titration calorimetry (ITC), mass spectrometry and differential scanning fluorimetry (DSF) were used to characterise B. anthracis DHDPS and compare it with the well characterised Escherichia coli enzyme. B. anthracis DHDPS exhibited different kinetic behaviour compared with E. coli DHDPS, in particular, substrate inhibition by (S)aspartate semi-aldehyde was observed for the B. anthracis enzyme (Ksi(ASA) = 5.4 ± 0.5 mM), but not for the E. coli enzyme. As predicted from a comparison of the X-ray crystal structures, the B. anthracis enzyme was not inhibited by lysine. The B. anthracis enzyme was thermally stabilised by the first substrate, pyruvate, to a greater extent than its E. coli counterpart, but has a weaker affinity for pyruvate based on enzyme kinetics and ITC studies. This characterisation will provide useful information for the design of inhibitors as new antibiotics targeting B. anthracis. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the causative agent of anthrax disease, which often results in severe respiratory, gastrointestinal or cutaneous infections [1,2]. Although the disease most commonly infects livestock, particularly in developing countries, it can be transmitted to humans through contact with sick animals, infected animal products, or through inhalation of the spores [2]. Given that the spores are easy to produce in the lab and highly resistant to UV light, extreme temperatures and pH and high salinity levels, as well as routine disinfection methods [3], the use of B. anthracis in bioterrorism and biological warfare is recognised as a threat to society. In 2001, for example, a number of letters containing B. anthracis spores were distributed via the U.S. postal system. This

Abbreviations: ASA, (S)-aspartate semi-aldehyde; DAP, meso-diaminopimelate; DHDPR, dihydrodipicolinate reductase; DHDPS, dihydrodipicolinate synthase; ITC, isothermal titration calorimetry; DSF, differential scanning fluorimetry; dRFU/dt, change in fluorescence (relative fluorescence units) with respect to time ⁎ Corresponding authors. Fax: +64 3 364 2590. E-mail addresses: [email protected] (J.A. Gerrard), [email protected] (S.R.A. Devenish). 1570-9639/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2009.06.020

intentional act resulted in 11 cases of respiratory anthrax and the death of 5 innocent citizens, although as many as 30,000 people may have been exposed to the spores [1]. It also resulted in significant panic amongst affected communities and demonstrated the vulnerability of Western countries to bioterrorism. In the time since, there has been heightened interest in the development of anti-anthrax agents with most focus on establishing a safe and effective vaccine [1]. Although there is a suitable anti-anthrax vaccine available, there are well documented issues with its challenging production, its safety amongst users, and speed of delivery, particularly to military personnel [1]. Anthrax can also be treated using antibiotics. The first antibiotic used against the disease was penicillin and since then a number of different antibiotic compounds have been deployed [4]. However, the emergence of antibioticresistant strains of B. anthracis has meant that most antibiotics need to be administered as part of a cocktail [2]. Along with these issues comes the fear that future terrorist attacks will involve engineered antibiotic-resistant strains of B. anthracis [4]. Accordingly, there is an urgent need to discover new antibiotics for rapid and effective treatment of anthrax and an equally urgent need to characterise new drug targets. One such drug target is dihydrodipicolinate synthase (DHDPS).

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DHDPS catalyses the first committed step in the biosynthetic pathway, yielding the essential amino acid, lysine (Fig. 1) [5]. Lysine, and its precursor meso-diaminopimelic acid (DAP), are integral components of the bacterial cell wall providing cross-linking and enhanced turgor rigidity. Dipicolinic acid, a product derived from HTPA via oxidation, is involved in generation of the heat resistant resting-state of the Gram-positive endospore, constituting N10% of the total mass [6]. These dual pathways make DHDPS a potential antibiotic target in microbes [7]. Mammals do not synthesise lysine, but acquire it from dietary sources, which means that antibiotics targeting DHDPS are not expected to show mammalian toxicity. Furthermore, bacterial DHDPS has been demonstrated to be the product of an essential gene [8] making DHDPS a valid, but as yet uncharted, antibiotic target [9]. DHDPS catalyses the condensation of pyruvate and (S)-aspartate semi-aldehyde (ASA) to form (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid (HTPA) as shown in Fig. 1 [10]. The mechanism of DHDPS has been studied extensively, particularly for the E. coli enzyme [10,11], and has been shown to have a ping-pong kinetic mechanism: the first substrate (pyruvate) binds, followed by release of water, and then the second substrate (ASA) binds, and the product is released. It had been suggested that high concentrations of ASA inhibited DHDPS from E. coli [12], but this was later shown to be merely an artefact of the ASA preparation [13]. Furthermore, it has been demonstrated that DHDPS is feedback inhibited by lysine, the end product of the pathway [9,12,14]. Comparatively, DHDPS from plants is strongly inhibited by lysine, DHDPS enzymes from Gram-negative bacteria, such as E. coli, are weakly inhibited by lysine, but physiological concentrations of lysine have not been shown to inhibit DHDPS from Gram-positive bacteria to date [15–17] and thus lysine inhibition was predicted to be absent from the B. anthracis enzyme. DHDPS from most species characterised so far: e.g. E. coli, PDB ID 1dhp [18] and 1yxc [19], Agrobacterium tumefaciens, PDB 2hmc, Thermotoga maritima, PDB 1o5k [15], Mycobacterium tuberculosis, PDB 1xxx [16], Aquifex aeolicus, PDB 2ehh, Nicotiana sylvestris, published but not available from PDB [20], Hahella chejuensis, PDB 2rfg, Neisseria meningitidis PDB 3flu [21], Corynebacterium glutamicum, PDB 3cpr [17], and B. anthracis, PBD 1xky [22] are homotetramers, with each monomer consisting of a (β/α)8-barrel adorned with three extra αhelices at the C-terminus of the chain [23]. Each monomer contains one active site, which is situated in the centre of the barrel [18]. There is also a lysine binding site located in the cleft at the tight dimer interface with one lysine molecule binding per monomer [14]. Comparison of the crystal structures shows that while all are

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homotetramers, different quaternary architectures exist for bacterial and plant enzymes [20,24]. This, coupled with the two recent papers [25,26] showing that the structure of DHDPS from Staphylococcus aureus is, intriguingly, a dimer, has promoted interest in the evolution of quaternary structure in this enzyme [24,25,27]. The BA3935 gene of B. anthracis, referred to as dapA2, encodes a 292 amino acid protein with a subunit molecular weight of 31,233 Da. This protein was identified as dihydrodipicolinate synthase (DHDPS) by comparison with DHDPS from other organisms [22]. A second gene in B. anthracis (BA2832, dapA1) has 32% sequence identity with dapA2 [22], but as yet has not been biochemically verified as a DHDPS. The crystal structure of DHDPS from B. anthracis (BA3935, dapA2) has been determined [22], and shows strong overall structural similarity to DHDPS from E. coli, with which it shares 43% sequence identity. The DAP/lysine biosynthetic pathway is thought to be of particular importance in Gram-positive bacteria given the presence of a thicker cell wall, in which DAP makes up a higher proportion of the dry weight compared with Gram-negative bacteria [22]. Additionally, 10% of the dry weight of B. anthracis spores is dipicolinate, which is formed by the further oxidation of HTPA, the product of the DHDPS reaction [22]. This further validates B. anthracis DHDPS as a novel antibiotic target. In this paper, we report a kinetic characterisation of DHDPS from B. anthracis (Ba-DHDPS), and compare the enzyme with the better characterised DHDPS from E. coli (Ec-DHDPS). We also confirm that there is no allosteric regulation of the enzyme by lysine and report preliminary data on the stability of the enzyme, examined by isothermal titration calorimetry (ITC) and differential scanning fluorimetry (DSF). Taken together, these data inform our attempts to inhibit the enzyme as a first step in antibiotic design. 2. Materials and methods 2.1. Materials Chemicals were purchased from the Sigma-Aldrich Co., Codexis or Invitrogen, unless stated otherwise. 2.2. Expression and purification The plasmid pB3935 [22] contains a His-tagged copy of the dapA2 gene from B. anthracis Ames and a gene conferring kanamycin resistance. The pB3935-dapA2 clone was transformed into E. coli BL21(DE3) for protein over-expression and plated out on Luria–Bertani (LB) medium with kanamycin (30 μg/mL) and

Fig. 1. The reaction catalysed by DHDPS [10], and important products of the lysine biosynthetic pathway.

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glucose (0.5%). Plates were grown overnight at 37 °C. A single colony was then transferred to 10 mL LB broth, containing kanamycin (30 μg/mL) and glucose (0.5%) and grown at 30 °C with shaking for 3 h. The culture was then transferred into 800 mL of ZYM5052 auto-inducing media [28], grown at 37 °C with shaking for 3 h, then transferred to room temperature overnight. To harvest cells, the culture was centrifuged using an Eppendorf centrifuge 5810R for 20 min at 3200 g and the supernatant discarded. The pellet was washed in phosphate buffer (20 mM Na2HPO4, 100 mM NaCl, pH 8.0) and centrifuged for 10 min at 3200 g. The cells were then resuspended in Buffer A (20 mM Na2HPO4, 0.5 M NaCl, 30 mM imidazole, pH 8) and lysed by sonication on ice using a Sonics Vibra Cell sonicator (50 W, 5 min total on, pulsing 1 s on, 5 s off). The cell free supernatant was collected by centrifugation at 18,500 g for 20 min. The crude protein was then loaded onto a GE Healthcare His-Trap FF Crude 5 mL column pre-equilibrated with Buffer A. After the protein was loaded, the column was washed with Buffer A and then eluted with Buffer B (20 mM Na2HPO4, 0.5 M NaCl, 500 mM imidazole, pH 8). The protein containing fractions were pooled and dialysed into 200 mM HEPES, pH 8. The concentration of the purified protein was determined using A280 measurements obtained on a NanoDrop ND1000 spectrometer and the extinction coefficient of 0.2775 AU/(mg/mL)·cm calculated with Protein Calculator v. 3.3 (www.scripps.edu/~edputnam/protcalc.html).

OriginPro v8.0724 (OriginLab Corporation), fitting data to the pingpong with uncompetitive substrate inhibition (Eq. (1)) and pingpong with competitive substrate inhibition models (Eq. (2)) [30]. Assays were also performed using Ec-DHDPS, according to previously published methods [29] (1 μg/mL Ec-DHDPS, 50 μg/mL Ec-DHDPR, 0.2 mM NADPH and varying pyruvate and ASA concentrations in 100 mM HEPES, pH 8.0). initial rate = Vmax
½A
½B   = KMðAÞ
½B + KMðBÞ
½A + ½A
½B
ð1 + ½B = Ksi Þ :

ð1Þ

initial rate = Vmax
½A
½B   = KMðAÞ
½B
ð1 + ½B = Ksi Þ + KMðBÞ
½A + ½A
½B :

ð2Þ

In Eqs. (1) and (2), Vmax is the maximal velocity, [A] and [B] are the concentrations of pyruvate and ASA respectively, KM(A) and KM(B) are the Michaelis–Menten constants for pyruvate and ASA respectively, and Ksi is the equilibrium dissociation constant for inhibition by the substrate ASA. Potential feedback inhibition was examined by the addition of the amino acids lysine, diaminopimelate (DAP), threonine, methionine and isoleucine. These were added to the assay (conditions as described above, with pyruvate fixed at 10 mM and ASA at 0.5 mM) at 1 mM, 10 mM and 100 mM concentrations and the percentage of maximum activity was recorded.

2.3. Liquid chromatography mass spectrometry 2.5. Isothermal titration calorimetry (ITC) Mass spectrometric data were collected using an Agilent 6510 LC/ Q-TOF mass spectrometer with an electrospray ionising (ESI) source coupled to an Agilent 1100 LC system (Agilent, Palo Alto, CA). The enzyme was injected directly (5 μL) into the mass spectrometer with 25% v/v acetonitrile in water and 0.1% v/v formic acid at 0.25 mL/min. All data were acquired and reference mass corrected via a dual-spray ESI source. Each scan or data point on the total ion chromatogram was an average of 10,000 transients, producing a scan every second. Spectra were created by averaging the scans across each peak. The conditions for the mass spectrometer were: positive mode; drying gas flow was 7 L/min; nebuliser was 30 psi; drying gas temp was 325 °C; Vcap was set to 4000 V; the fragmentor was set to 225 V; the skimmer was set to 60 V; the OCT RFV was 750 V; and the scan range acquired was 100–2500 m/z. 2.4. Kinetic analysis A coupled assay using dihydrodipicolinate reductase (DHDPR) from E. coli prepared by previously published methods was used for kinetic analysis [29]. The reaction was initiated in a thermally equilibrated 1 mL cuvette by the addition of DHDPS. The change in absorbance at 340 nm was recorded over 120 s at 30 °C, blanked against dH2O. Controls were performed to ensure that DHDPR was saturating and that reaction rate was proportional to the concentration of DHDPS. Assays were carried out in duplicate or triplicate, with error typically within 10%. The effect of pH on the initial rate of Ba-DHDPS was measured using 200 mM HEPES buffer from pH 7.35 to pH 8.55 and using 200 mM Bis–Tris propane buffer from pH 7.0 to 9.5. The effect of buffer concentration on the rate of Ba-DHDPS was measured using 50 mM, 100 mM and 200 mM concentrations of Bis–Tris propane buffer. Kinetic analysis of Ba-DHDPS was performed by measuring the initial rate while varying concentrations of pyruvate (0.05–5 mM) and ASA (0.05–5 mM). The concentration of Ba-DHDPS used in the assay was 1.08 μg/mL, while DHDPR was 50 μg/mL, and the assay buffer used for full kinetic characterisation was 200 mM Bis–Tris propane, pH 8.5. Kinetic parameters were determined using

ITC was performed on a MicroCal VP-ITC unit using methods based on those outlined by Turnbull and Daranas [31]. All experiments were conducted at 20 °C and solutions were degassed under vacuum prior to use. The protein concentration used was 1.95 mg/mL, corresponding to a monomer concentration of 0.06 μmol/mL (Ba-DHDPS MW 32,287 Da). A 35 injection protocol was used in which ligand was titrated (1 × 1 μL, 20 × 5 μL then 15 × 10 μL) at 210 second intervals into the Ba-DHDPS protein solution. The concentrations of ligands used were 2 mM sodium pyruvate, 10 mM lysine, 2 mM ASA, which were made up in the stock buffer solution of 200 mM HEPES, pH 7.7. Heats of dilution determined in the absence of protein were subtracted from the titration data prior to curve fitting. Additionally, an initial 1 μL injection was discarded from each dataset in order to remove the effect of titrant diffusion across the syringe tip during the equilibration process. Curve fitting was undertaken in Origin v5.0 using the standard non-interacting one site model supplied by MicroCal. From this, reaction stoichiometry (n), dissociation binding constant (Kd), enthalpy (ΔH) and entropy (ΔS) were calculated. For comparison, EcDHDPS was run at 0.055 μmol/mL titrated with 2 mM pyruvate under exactly the same injection regime. Reported values are the averages of at least two independent trials ± the standard error. 2.6. Differential scanning fluorimetry (DSF) DSF was performed using the BioRad IQ5 Multicolor Real-Time PCR Detection System, with methods based on those of Niesen et al. [32]. A plate was set up with each well containing protein (4 μL, 1.5 mg/mL), SYPRO orange dye (1 μL) and buffer (20 μL HEPES (200 mM, pH 8) or Bis-Tris propane (200 mM, pH 8.5) and potential ligands: pyruvate (10 mM, 50 mM); methionine (10 mM, 50 mM); threonine (10 mM, 50 mM); DAP (10 mM, 50 mM); isoleucine (10 mM, 50 mM); lysine (10 mM, 50 mM)). A control well containing HEPES (200 mM, pH 8) and dye, but no protein, was also set up and used as a blank for all measurements. Temperature was stepped from 20 °C to 94 °C in 0.3 °C steps. Each temperature was held for 20 s prior to measuring and unfolding temperature was determined as the point of maximum inflection of the dRFU/dt curve.

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3. Results and discussion

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Table 2 Percent of full activity for Ba-DHDPS and Ec-DHDPS in the presence of a number of different potential feedback inhibitors at varying concentrations.

3.1. Mass spectrometry

% activity

Ba-DHDPS was purified to homogeneity using published methods. Denaturing ESI-TOF mass spectrometry of the enzyme showed a peak at 32,287 Da, consistent with the predicted mass of the His-tagged protein (Fig. S1).

L-lysine

DAP

3.2. Enzyme kinetics L-methionine

Based on the similarity of the active sites of Ba-DHDPS and Ec-DHDPS, the same mechanism and kinetic model were expected for the two enzymes. Kinetic analysis was carried out using the coupled assay with NADPH-dependent DHDPR [33], the next enzyme in the DAP pathway. The effect of pH on rate was measured in order to find the optimal pH for full kinetic characterisation. The observed rate of Ba-DHDPS with varying pH showed that the rate increased until it reached a maximum at pH 8.5, from which it steadily decreased (data not shown). The maximal rate was obtained in 200 mM Bis–Tris propane buffer, so this buffer at pH 8.5 was used for all further assays. The kinetic parameters of Ba-DHDPS were determined through analysis of the initial rates at varying concentrations of both of the substrates, ASA and pyruvate (Figs. S2 and S3). The results were analysed using OriginPro 8 to estimate kinetic parameters and found to be similar to previously published values for Ec-DHDPS (Table 1). The KM for both substrates was higher in Ba-DHDPS than the E. coli counterpart. Intriguingly, Ba-DHDPS exhibited uncompetitive substrate inhibition by ASA at high concentrations (Ksi(ASA) = 5.4 ± 0.5 mM), something that is not observed for DHDPS from Ec-DHDPS. Dobson et al. note that substrate inhibition observed in early studies of Ec-DHDPS was an artefact of preparations of ASA synthesised by ozonolysis; thus when ASA was prepared by a different route, no substrate inhibition was observed [13]. In order to ensure that this was not the case for the ASA inhibition observed for Ba-DHDPS, kinetic measurements were performed for Ec-DHDPS using the same batch of ASA that was used in the Ba-DHDPS assays. No substrate inhibition was observed for Ec-DHDPS, lending confidence to the substrate inhibition observed for the B. anthracis enzyme not being artefactual. It is not unusual for ping-pong enzymes, like DHDPS [12], to display competitive inhibition by the second substrate, caused by premature binding of this substrate before the first substrate. Recently, DHDPS from N. meningitidis was found to be competitively inhibited by ASA [21], but the inhibition in the case of Ba-DHDPS was clearly not of the competitive type, as evidenced by the fact that increasing ASA concentration caused minimal change in the apparent KM(PYR), but clearly impacted the maximal rate, and the very poor fit of the competitive type model to the data (adjusted R2 for the fit of 0.904, Figs. S4 and S5). The uncompetitive substrate inhibition model gave a superior, albeit not perfect fit to the data (adjusted R2 for the fit of 0.966). This inhibition model implies that the inhibitory binding of ASA takes place after the pyruvate has already bound in the active site, and could be prior to formation of the Schiff base, causing inhibition of that critical step, or alternatively, a second molecule of ASA could bind to the Schiff base–ASA complex and inhibit progression of the

Table 1 Kinetic parameters from the characterisation of Ba-DHDPS, compared with Ec-DHDPS.

kcat (s-1) KM(ASA) (mM) KM(PYR) (mM) Ksi(ASA) (mM)

Ba-DHDPS

Ec-DHDPS [29]

76 ± 3 0.18 ± 0.02 0.43 ± 0.03 5.4 ± 0.5

124 ± 7 0.11 ± 0.01 0.25 ± 0.03 –

Errors are standard error of the mean for the global fit to triplicate rate measurements.

L-threonine

L-isoleucine

1 mM 10 mM 100 mM 1 mM 10 mM 100 mM 1 mM 10 mM 100 mM 1 mM 10 mM 100 mM 1 mM 10 mM 100 mM

Ba-DHDPS

Ec-DHDPS

100 ± 4 98 ± 6 88 ± 2 103 ± 4 99 ± 1 91 ± 2 96 ± 3 80 ± 2 80 ± 7 102 ± 1 89 ± 2 40 ± 1 98 ± 5 94 ± 3 90 ± 4

23 ± 3 19 ± 3 13 ± 3 108 ± 1 122 ± 3 118 ± 6 99 ± 1 112 ± 2 111 ± 5 96 ± 1 84 ± 2 33 ± 1 97 ± 5 97 ± 3 94 ± 3

Measurements were performed in triplicate and error is shown as the standard deviation.

reaction. This model of inhibition has been previously observed in the ping-pong enzyme quinohaemoprotein ethanol dehydrogenase type 1 from Comamonas testosteroni [30], but is otherwise uncommon, suggesting that analogues of ASA may be suitable lead compounds for future antibiotic development efforts specific to B. anthracis. 3.3. Feedback inhibition Feedback inhibition of Ec-DHDPS by lysine has been well characterised [12] and is an important regulatory mechanism for controlling flux. In order to investigate the effect of potential feedback inhibitors or effectors on the initial rate of Ba-DHDPS, enzyme kinetic measurements were repeated in the presence of lysine, DAP, methionine, threonine and isoleucine. These amino acids were chosen due to their positions in the biosynthetic pathway involving DHDPS, and therefore their potential as regulators of the enzyme. Sequence alignment with Ec-DHDPS revealed that, like the lysine insensitive DHDPS from C. glutamicum [17], Ba-DHDPS lacked the three key lysine binding residues H53, H56 and E84 (E. coli numbering), with these residues being replaced in Ba-DHDPS with serine, lysine and alanine respectively. DHDPS enzymes from Grampositive bacteria show little or no feedback inhibition by lysine [16,34], therefore none was expected for Ba-DHDPS, consistent with analysis of the crystal structure. The results confirmed this hypothesis (Table 2) and found no evidence of inhibition by lysine or any of the other molecules tested at physiological concentrations. 3.4. Differential scanning fluorimetry (DSF) DSF is a technique that is used to identify ligands that bind and stabilise purified proteins [32]. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye, in this case SYPRO orange, which has an affinity for hydrophobic regions of the protein and which fluoresces in a hydrophobic environment but not a hydrophilic one [32]. As the protein unfolds, the hydrophobic regions are exposed and the fluorescence therefore increases. Ligands that bind and stabilise purified proteins will cause an increase in the unfolding temperature as measured by DSF. DSF showed that BaDHDPS and Ec-DHDPS have similar thermostability, unfolding at 59.8 °C and 61.3 °C respectively. The effect of different ligands on the thermal stability of Ba-DHDPS and Ec-DHDPS was investigated. Specifically, the ligands used were the substrate pyruvate, and the amino acids tested for feedback inhibition in the kinetic analysis. Pyruvate has previously been found to stabilise DHDPS from Ba-DHDPS and other species [16,23,35,36]. No

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DHDPS but not Ba-DHDPS (Fig. 2), consistent with the inhibition data above. The substrate pyruvate is known to form a Schiff base with the active site residue lysine 161 (E. coli numbering) [10], but the reason for the stabilisation being greater for Ba-DHDPS than for the E. coli enzyme was unclear, and therefore warranted further investigation by ITC. 3.5. Isothermal titration calorimetry (ITC)

Fig. 2. Change in unfolding temperature of Ba-DHDPS (light) and Ec-DHDPS (dark) as measured by DSF in the presence of the indicated ligands at 10 mM and 50 mM concentrations (Pyr = pyruvate, Met = methionine, Thr = threonine, DAP = diaminopimelate, Iso = isoleucine and Lys = lysine). Measurements were performed in triplicate and the error bars represent the standard deviation.

noteworthy change in unfolding temperature was seen for methionine, threonine, DAP or isoleucine (Fig. 2). This result is consistent with the results of the kinetic analysis. Pyruvate was seen to increase the unfolding temperature of both enzymes, although Ba-DHDPS was stabilised to a greater degree (Fig. 2). Lysine was seen to stabilise Ec-

ITC is uniquely able to quantify and discriminate between enthalpic and entropic contributions to binding and it serves as a crucial bridge between experimental data and computational approaches to the analysis of ligand binding [37]. Furthermore, since thermodynamic parameters can be determined in the absence of catalytic turnover, ITC can also serve to elucidate aspects relating to the mechanism of catalysis and allosteric control otherwise inaccessible via conventional kinetic assays. Recently, an ITC investigation of MosA, a DHDPS from Sinorhizobium meliloti, characterised the binding thermodynamics with respect to pyruvate, the pyruvate analogue 2oxobutyrate and the allosteric inhibitor lysine [38,39]. We carried out ITC experiments to examine the binding interactions of Ba-DHDPS with pyruvate, ASA and lysine. Comparative experiments were also conducted with Ec-DHDPS to enable comparison of the derived thermodynamic parameters. The raw ITC binding data for the titration of pyruvate into DHDPS for both B. anthracis and E. coli generated hyperbolic isotherms, confirming that both enzymes were weak binders of pyruvate (Fig. 3). The titrant concentration and injection regime were optimised to ensure adequate coverage of the whole isotherm within a single experiment. A beneficial aspect of this optimisation is that all

Fig. 3. ITC titrations of pyruvate into Ba-DHDPS (0.06 mM, A) and Ec-DHDPS (0.055 mM, B) following titration baseline subtraction. The top graph shows the raw data for 35 injections of 2 mM pyruvate into a solution of DHDPS in 200 mM HEPES at 20 °C. The middle graph data points show integrated heats of interaction as a function of molar ratio, and the solid line represents the line of best fit obtained for a single binding site model. Residuals were calculated as the difference between the line of best fit and the experimental values. Note: the increases in exothermicity at 75 min in the top graph is due to an increased titre volume.

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parameters of the binding algorithm can be left floating. Whilst it has been suggested that low affinity systems necessitate the constraining of n to a value consistent with known binding stoichiometry [31,40] a recent statistical analysis indicates that freezing n is not necessary, provided certain criteria are met [41]. Modelling of the titration isotherm using a single independent binding model resulted in a calculated Kd of 0.0186 ± 0.0003 mM and 0.0299 ± 0.0005 mM for B. anthracis and E. coli respectively (Table 3). The quality of the fit obtained from least squares regression implies that each binding site within the homotetrameric DHDPS is identical and independent, which is consistent with previous literature on the E. coli enzyme [29], or that they have Kd values indistinguishable at the resolution provided for by ITC. Attempts to constrain n = 1 (Fig. S6) or to fit other binding models (two binding sites, sequential binding sites and multiple binding sites) gave poor fits to the experimental data. The thermodynamic parameters for pyruvate binding to BaDHDPS and Ec-DHDPS are very similar, with calculated ΔG values of −26.6 kJ/mol and −26.09 kJ/mol respectively; however, significant differences exist between the values reported here and those determined for the enzyme isolated from MosA [38] (Table 3). A detailed investigation of the E. coli enzyme to explore this discrepancy will be reported elsewhere. Whilst the Kd value determined for MosA more closely reflects the kinetically determined value of KM(PYR) (0.27 ± 0.02 mM, [42]), it should be noted that Kd and KM represent fundamentally different phenomena, specifically the substrate dissociation constant of the ligand from the macromolecular complex in the absence of catalysis (Kd) and the steady state dissociation constant for the productive enzyme complex determined from initial velocities studies under steady state catalysis (KM) [43]. The contribution from individual enthalpy (ΔH) and entropy (ΔS) components to the overall free energy (ΔG) are also significantly different. The interaction of MosA with pyruvate was characterised as being entropically governed [38], whereas our best fit parameters indicate the process to be enthalpically driven for B. anthracis and E. coli, with entropy making a relatively slight contribution (Table 3). The binding isotherm modelled for B. anthracis indicates that the exothermic reaction is primarily governed by a favourable enthalpic contribution (−35.2± 0.4 kJ/mol), presumably arising from Schiff base formation between pyruvate and the active site lysine, which overcomes the unfavourable entropic effect of binding (−8.6 ± 0.2 kJ/mol). A similar thermodynamic profile is observed for E. coli, although in this instance a greater enthalpic component is balanced by a greater entropic value (−42.5 ± 0.6 kJ/mol versus −16.4 ± 0.3 kJ/mol) resulting in a comparable total free energy for the binding interaction. A relatively small entropic component arising from changes in the conformational entropy is anticipated for the interaction of DHDPS with pyruvate. X-ray crystallographic studies indicate that no significant protein rearrangements accompany the binding of pyruvate to the active site of Ec-DHDPS [19,44]. Consequently, it is posited that the entropy change observed for the pyruvate-DHDPS binding interaction arises primarily from changes in the relative hydration of

Table 3 Thermodynamic data for pyruvate binding to Ba-DHDPS, Ec-DHDPS and MosA.

N Kd (mM) ΔH (kJ/mol) ΔS (J/mol/deg) TΔS (kJ/mol) ΔG (kJ/mol) c-value

B. anthracis

E. coli

MosA

0.637 ± 0.005 0.0186 ± 0.0003 − 35.2 ± 0.4 − 29.5 ± 0.5 − 8.6 ± 0.2 − 26.6 ± 0.2 2.06

0.68 ± 0.001 0.02985 ± 0.0005 − 42.5 ± 0.6 − 56.1 ± 0.9 − 16.4 ± 0.3 − 26.09 ± 0.05 1.5

1 0.4 ± 0.1 − 3.8 ± 0.6 53.7 ± 3.4 16 ± 1 − 19.8 ± 0.5 0.25

Experiments were conducted at 20 °C and analysed using the single independent site binding model (MicroCal VP-ITC, Origin 5.0) with ΔH, K and n floating. Values are an average of at least two independent trials ± standard error. The reported MosA values were collected at 25 °C and analysed with a fixed n = 1 [38].

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the charged pyruvate ligand relative to the bulk solvent and from increased ordering of water molecules within the active site and surrounding hydration shell. As previously noted, caution must be applied in obtaining ITC data of low affinity interactions, and in the subsequent modelling and interpretation of the resultant isotherm [31,40]. Adequate coverage of data points, sampled at both low and high ligand to protein ratios, is necessary to provide a reliable determination of the dissociation constant Kd, which in turn directly affects the calculated values of ΔH and ΔS. Constraining the binding stoichiometry to n = 1 also contributes significant variance to the fitted parameters of Kd (Fig. S6). Isothermal titration experiments were also conducted on Ba-DHDPS with the second substrate ASA and with lysine, the allosteric inhibitor of Ec-DHDPS. Consistent with the kinetic characterisation of a ping-pong mechanism with uncompetitive inhibition by ASA, and the DSF ligand binding experiments, no significant exothermic reaction was observed for either of these ligands (Fig. S7). 4. Conclusions The search for effective treatments for the mammalian disease anthrax leads to the investigation of enzymes that have a key role in the survival of the causative bacteria, B. anthracis [22]. Most recent research has focused on the enzymes involved in the toxin system of the bacteria; however, as there still remains much to be understood regarding this system, the development of new effective antibiotics targeting enzymes outside this system is necessary [45]. The enzyme DHDPS is an antibiotic target due to its key role in the lysine/DAP biosynthetic pathway [5], and this characterisation of DHDPS from B. anthracis provides further insight into the action of this essential enzyme. Despite showing significant structural similarity to its E. coli counterpart, Ba-DHDPS demonstrated substrate inhibition by the second substrate, ASA (Ki(ASA) = 5.4 ± 0.5 mM). This observed substrate inhibition raises questions regarding the regulation of DHDPS in B. anthracis, and also suggests new avenues of inhibitor design, based on ASA, that may show specificity for B. anthracis. As was predicted by examination of the lysine binding site of DHDPS from B. anthracis, lysine was not a feedback inhibitor of BaDHDPS. A number of other amino acids were also screened as potential allosteric inhibitors of DHDPS, although none were shown to inhibit at a physiologically relevant concentration. These same amino acids, including lysine, were also investigated through DSF for their ability to thermally stabilise DHDPS from B. anthracis. Lysine was seen to stabilise Ec-DHDPS but not Ba-DHDPS, a result consistent with the lack of feedback inhibition by lysine. Using DSF, pyruvate was shown to stabilise both DHDPS from E. coli and B. anthracis, although Ba-DHDPS was stabilised to a larger extent. How this observation relates to the results of the ITC experiment warrants further investigation. However, we suggest that the higher affinity of binding of pyruvate in Ba-DHDPS facilitates the greater stability observed. The thermodynamic characterisation of Ba-DHDPS with ITC revealed that the pyruvate binding interaction was exothermic and favourable (ΔG −26.6 ± 0.2 kJ/mol) but with low affinity (Kd = 0.0186 ± 0.0003 mM). Fitting of these data to a single independent binding model indicates that the pyruvate interaction is primarily driven by a favourable enthalpic component (ΔH −35.2 ± 0.4 kJ/mol), presumably due to Schiff base formation, and is opposed by a minor entropy effect (TΔS = − 8.6 ± 0.2 kJ/mol). These values are similar to the binding parameters determined for Ec-DHDPS. In summary, this study has biochemically characterised DHDPS from B. anthracis, revealed new starting points for the design of novel antibiotics for B. anthracis, and highlights the need to examine enzymes from pathogens, rather than rely on data from model organisms.

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Acknowledgements We would like to acknowledge the Defense Threat Reduction Agency (DTRA) (DTRA Project ID AB07CBT004) and the Australian Research Council for providing an Australian Postdoctoral Fellowship for MAP. ACMT is grateful to the Growth and Innovation Pilot Initiative for postdoctoral funding, and SRAD thanks the Foundation for Research, Science and Technology for postdoctoral funding (contract UOCX0603). RCJD acknowledges the C.R. Roper bequest for support. MJF is currently funded by the European Commission as part of BaSysBio (Bacillus Systems Biology), contract no. LSHG-CT-2006037469 under the 6th framework program “Priority 1 Life Sciences, Genomics and Biotechnology for Health”. The authors thank Jackie Healy for voluptuous technical support, Marie Squire for mass spectrometry, and Grant Pearce for formidable discussion. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbapap.2009.06.020. References [1] R.N. Brey, Molecular basis for improved anthrax vaccines, Adv. Drug Deliv. Rev. 57 (2005) 1266–1292. [2] S. Oncu, S. Oncu, S. Sakarya, Anthrax — an overview, Med. Sci. Monit. 9 (2003) RA276–RA283. [3] A. Watson, D. Keir, Information on which to base assessments of risk from environments contaminated with anthrax spores, Epidemiol. Infect. 113 (1994) 479–490. [4] L.W.J. Baillie, Past, imminent and future human medical countermeasures for anthrax, J. Appl. Microbiol. 101 (2006) 594–606. [5] Y. Yugari, C. Gilvarg, The condensation step in diaminopimelate synthesis, J. Biol. Chem. 240 (1965) 4710–4716. [6] M. Paidhungat, B. Setlow, A. Driks, P. Setlow, Characterization of spores of Bacillus subtilis which lack dipicolinic acid, J. Bacteriol. 182 (2000) 5505–5512. [7] C.A. Hutton, T.J. Southwood, J.J. Turner, Inhibitors of lysine biosynthesis as antibacterial agents, Mini-Rev. Med. Chem. 3 (2003) 115–127. [8] K. Kobayashi, S.D. Ehrlich, A. Albertini, G. Amati, K.K. Andersen, M. Arnaud, K. Asai, S. Ashikaga, S. Aymerich, P. Bessieres, F. Boland, S.C. Brignell, S. Bron, K. Bunai, J. Chapuis, L.C. Christiansen, A. Danchin, M. Debarbouille, E. Dervyn, E. Deuerling, K. Devine, S.K. Devine, O. Dreesen, J. Errington, S. Fillinger, S.J. Foster, Y. Fujita, A. Galizzi, R. Gardan, C. Eschevins, T. Fukushima, K. Haga, C.R. Harwood, M. Hecker, D. Hosoya, M.F. Hullo, H. Kakeshita, D. Karamata, Y. Kasahara, F. Kawamura, K. Koga, P. Koski, R. Kuwana, D. Imamura, M. Ishimaru, S. Ishikawa, I. Ishio, D. Le Coq, A. Masson, C. Mauel, R. Meima, R.P. Mellado, A. Moir, S. Moriya, E. Nagakawa, H. Nanamiya, S. Nakai, P. Nygaard, M. Ogura, T. Ohanan, M. O'Reilly, M. O'Rourke, Z. Pragai, H.M. Pooley, G. Rapoport, J.P. Rawlins, P. Stragier, R. Studer, H. Takamatsu, T. Tanaka, M. Takeuchi, H.B. Thomaides, V. Vagner, J.M. van Dijl, K. Watabe, A. Wipat, H. Yamamoto, M. Yamamoto, Y. Yamamoto, K. Yamane, K. Yata, K. Yoshida, H. Yoshikawa, U. Zuber, N. Ogasawara, Essential Bacillus subtilis genes, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 4678–4683. [9] C.A. Hutton, M.A. Perugini, J.A. Gerrard, Inhibition of lysine biosynthesis: an evolving antibiotic strategy, Mol. BioSystems 3 (2007) 458–465. [10] S. Blickling, C. Renner, B. Laber, H.D. Pohlenz, T.A. Holak, R. Huber, Reaction mechanism of Escherichia coli dihydrodipicolinate synthase investigated by X-ray crystallography and NMR spectroscopy, Biochemistry 36 (1997) 24–33. [11] R.C.J. Dobson, K. Valegård, J.A. Gerrard, The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad, J. Mol. Biol. 338 (2004) 329–339. [12] W.E. Karsten, Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by lysine, Biochemistry 36 (1997) 1730–1739. [13] R.C.J. Dobson, J.A. Gerrard, F.G. Pearce, Dihydrodipicolinate synthase is not inhibited by its substrate, (S)-aspartate β-semialdehyde, Biochem. J. 377 (2004) 757–762. [14] S. Blickling, J. Knablein, Feedback inhibition of dihydrodipicolinate synthase enzymes by (L)-lysine, Biol. Chem. 378 (1997) 207–210. [15] F.G. Pearce, M.A. Perugini, H.J. McKerchar, J.A. Gerrard, Dihydrodipicolinate synthase from Thermotoga maritima, Biochem. J. 400 (2006) 359–366. [16] G. Kefala, G.L. Evans, M.D. Griffin, S.R.A. Devenish, F.G. Pearce, M.A. Perugini, J.A. Gerrard, M.S. Weiss, R.C.J. Dobson, Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis, Biochem. J. 411 (2008) 351–360. [17] E.A. Rice, G.A. Bannon, K.C. Glenn, S.S. Jeong, E.J. Sturman, T.J. Rydel, Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein, Arch. Biochem. Biophys. 480 (2008) 111–121.

[18] C. Mirwaldt, I. Korndorfer, R. Huber, The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 Å resolution, J. Mol. Biol. 246 (1995) 227–239. [19] R.C.J. Dobson, M.D.W. Griffin, G.B. Jameson, J.A. Gerrard, The crystal structures of native and (S)-lysine-bound dihydrodipicolinate synthase from Escherichia coli with improved resolution show new features of biological significance, Acta Crystallogr. Sect. D 61 (2005) 1116–1124. [20] S. Blickling, H.-G. Beisel, D. Bozic, J. Knablein, B. Laber, R. Huber, Structure of dihydrodipicolinate synthase of Nicotiana sylvestris reveals novel quaternary structure, J. Mol. Biol. 274 (1997) 608–621. [21] S.R.A. Devenish, F.H. Huisman, E.J. Parker, A.T. Hadfield, J.A. Gerrard, Cloning and characterisation of dihydrodipicolinate synthase from the pathogen Neisseria meningitidis, Biochim. Biophys. Acta 1794 (2009) 1168–1174. [22] V.L. Blagova, V. Levdikov, N. Milioti, M.J. Fogg, A.K. Kalliomaa, J.A. Brannigan, K.S. Wilson, A.J. Wilkinson, Crystal structure of dihydrodipicolinate synthase (BA3935) from Bacillus anthracis at 1.94 Å resolution, Proteins: Struct. Funct. Bioinform. 62 (2006) 297–301. [23] B.B.B. Guo, S.R.A. Devenish, R.C.J. Dobson, A.C. Muscroft-Taylor, J.A. Gerrard, The C-terminal domain of Escherichia coli dihydrodipicolinate synthase (DHDPS) is essential for maintenance of quaternary structure and efficient catalysis, Biochem. Biophys. Res. Commun. 380 (2009) 802–806. [24] M.D.W. Griffin, R.C.J. Dobson, F.G. Pearce, L. Antonio, A.E. Whitten, C.K. Liew, J.P. Mackay, J. Trewhella, G.B. Jameson, M.A. Perugini, J.A. Gerrard, Evolution of quaternary structure in a homotetrameric protein, J. Mol. Biol. 380 (2008) 691–703. [25] B.R. Burgess, R.C.J. Dobson, M.F. Bailey, S.C. Atkinson, M.D.W Griffin, G.B. Jameson, M.W. Parker, J.A. Gerrard, M.A. Perugini, Structure and evolution of a novel dimeric enzyme from a clinically important bacterial pathogen, J. Biol. Chem. 283 (2008) 27598. [26] T.S. Girish, E. Sharma, B. Gopal, Structural and functional characterization of Staphylococcus aureus dihydrodipicolinate synthase, FEBS Lett. 582 (2008) 2923–2930. [27] S.R.A. Devenish, J.A. Gerrard, The role of quaternary structure in (β/α)8-barrel proteins: evolutionary happenstance or a higher level of structure–function relationships? Org. Biomol. Chem. 7 (2009) 833–839. [28] F.W. Studier, Protein production by auto-induction in high-density shaking cultures, Prot. Expression Purif. 41 (2005) 207–234. [29] R.C.J. Dobson, M.D.W. Griffin, S.J. Roberts, J.A. Gerrard, Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate, Biochimie 86 (2004) 311–315. [30] A. Geerlof, J.J.L. Rakels, A.J.J. Straathof, J.J. Heijnen, J.A. Jongejan, J.A. Duine, Description of the kinetic mechanism and enantioselectivity of quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni in the oxidation of alcohols and aldehydes, Eur. J. Biochem. 226 (2004) 537–546. [31] W.B. Turnbull, A.H. Daranas, On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J. Am. Chem. Soc. 125 (2003) 14859–14866. [32] F.H. Niesen, H. Berglund, V. Masoud, The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability, Nature Protocols 2 (2007) 2212–2221. [33] C.V. Coulter, J.A. Gerrard, J.A.E. Kraunsoe, D.J. Moore, A.J. Pratt, Escherichia coli dihydrodipicolinate synthase and dihydrodipicolinate reductase: kinetic an inhibition studies of two putative herbicide targets, Pestic. Sci. 55 (1996) 887–895. [34] D.P. Stahly, Dihydrodipicolinate synthase of Bacillus licheniformis, Biochim. Biophys. Acta 191 (1969) 439–451. [35] S.M. Halling, D.P. Stahly, Dihydrodipicolinate synthase of Bacillus licheniformis. Quaternary structure, kinetics, and stability in the presence of sodium chloride and substrates, Biochim. Biophys. Acta 452 (1976) 580–596. [36] J.E. Voss, S.W. Scally, N.L. Taylor, C. Dogovski, M.R. Alderton, C.A. Hutton, J.A. Gerrard, M.W. Parker, R.C.J. Dobson, M.A. Perugini, Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate, Acta Cryst Sect. F 65 (2009) 188–191. [37] T. Wiseman, S. Williston, J.F. Brandts, L.N. Lin, Rapid measurements of binding constants and heats of binding using a new titration calorimeter, Anal. Biochem. 179 (1989) 131–137. [38] C.P. Phenix, K. Nienaber, P.H. Tam, L.T.J. Delbaere, D.R.J. Palmer, Structural, functional and calorimetric investigations of MosA, a dihydrodipicolinate synthase from Sinorhizobium meliloti L5-30, does not support involvement in rhizopine biosynthesis, ChemBioChem 9 (2008) 1591–1602. [39] C.P. Phenix, D.R.J. Palmer, Isothermal titration microcalorimetry reveals the cooperative and noncompetitive nature of inhibition of Sinorhizobium meliloti L5-30 dihydrodipicolinate synthase by (S)-lysine, Biochemistry 47 (2008) 7779–7781. [40] J. Tellinghuisen, Optimizing experimental parameters in isothermal titration calorimetry, J. Phys. Chem. B 109 (2005) 20027–20035. [41] J. Tellinghuisen, Isothermal titration calorimetry at very low c, Anal. Biochem. 373 (2008) 395–397. [42] P.H. Tam, C.P. Phenix, D.R.J. Palmer, MosA, a protein implicated in rhizopine biosynthesis in Sinorhizobium meliloti L5-30, is a dihydropicolinate synthase, J. Mol. Biol. 335 (2004) 393–397. [43] M. Dixon, E.C. Webb, Enzymes, 3rd EdLongman, London, 1979. [44] S.R.A. Devenish, J.A. Gerrard, G.B. Jameson, R.C.J. Dobson, The high-resolution structure of dihydrodipicolinate synthase from Escherichia coli bound to its first substrate, pyruvate, Acta Crystallogr. Sect. F 64 (2008) 1092–1095. [45] A.M. Friedlander, Tackling anthrax, Nature 414 (2001) 160–161.