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Acta Crystallographica Section D: Structural Biology logoLink to Acta Crystallographica Section D: Structural Biology
. 2022 Jun 8;78(Pt 7):846–852. doi: 10.1107/S2059798322005125

Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)-2-bromopropionate

Lilian Chooback a,*, Leonard N Thomas b, Nathan Blythe a, William Karsten b
PMCID: PMC9248844  PMID: 35775984

The crystal structure of dihydrodipicolinate synthase modified with (S)-2-bromopropionate at Lys161 is reported.

Keywords: crystallography, dihydrodipicolinate synthase, (S)-2-bromopropionate, lysine biosynthesis, enzyme structure, enzyme inhibition

Abstract

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine-biosynthetic pathway converting pyruvate and l-aspartate-β-semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)-2-bromopropionate inactivates the enzyme in a pseudo-first-order process. An initial velocity pattern indicates that (S)-2-bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)-2-bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml−1 DHDPS. The crystals diffracted to 2.15 Å resolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active-site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half-reaction of the ping-pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)-2-bromopropionate indicates the covalent adduct formed from (S)-2-bromopropionate leads to a rotation of about 180° of the β–δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate.

1. Introduction

Bacterial infections create a serious need for the development of new antibacterial drugs (Alanis, 2005 ▸; Spellberg et al., 2008 ▸; Voelz et al., 2010 ▸). One area of research has been focused on inhibition of the l-lysine-biosynthetic pathway in bacteria. The l-lysine-biosynthetic pathway is an attractive target for the development of antibacterial compounds owing to the presence of this pathway in bacteria but not in mammals. Consequently, limited toxicity to humans could be expected for inhibitors of the pathway. Lysine is a member of the aspartate family of amino acids, which also includes methionine, threonine and isoleucine. In general, there are two different pathways for the biosynthesis of lysine: the diaminopimelate (DAP) pathway and the α-aminoadipic acid (AAA) pathway. Fungi and euglenoids use the AAA pathway to synthesize l-lysine. The DAP pathway is found in all bacteria, blue-green algae, vascular plants and phycomycete fungi with biflagellated spores. Neither pathway is found in mammals. Dihydrodipicolinate synthase (DHDPS) catalyzes the first rate-limiting step of lysine biosynthesis via the DAP pathway and involves the condensation of aspartate-β-semialdehyde (ASA) and pyruvate to form dihydropicolinate (DHDP) (Yugari & Gilvarg, 1965 ▸; Shedlarski & Gilvarg, 1970 ▸). In this reaction, the first substrate, pyruvate, initially forms a Schiff base with Lys161 (Escherichia coli numbering), which subsequently loses a proton to form a stable enamine intermediate (Karsten, 1997 ▸; Karsten et al., 2021 ▸). The binding of ASA, the second substrate, leads to formation of the final product of the reaction, DHDP (Shedlarski & Gilvarg, 1970 ▸).

Both lysine and DAP are found in the peptidoglycan of bacterial cell walls. DAP, which is an intermediate in the lysine-biosynthetic pathway, is a component of short peptide bridges that cross-link peptidoglycan polymer chains in the bacterial cell walls (Bartlett & White, 1985 ▸). Penicillin, cephalosporin and glycopeptide drugs such as vancomycin kill bacteria by disrupting the cross-linking of peptidoglycan strands in bacterial cell walls (Glauner et al., 1988 ▸; Neu, 1992 ▸). Other antibiotics such as d-cycloserine (Lambert & Neuhaus, 1972 ▸) act by inhibiting enzymes involved in the biosynthesis of the cell-wall components themselves. Interference with the DAP pathway has been shown to be a lethal event leading to cell lysis (Meadow et al., 1957 ▸; Rhuland, 1957 ▸), and therefore the pathway has received attention as a potential target for the development of new antibiotics.

Attempts have been made by a number of researchers to develop inhibitors of enzymes in the lysine-biosynthetic pathway (Girodeau et al., 1986 ▸; Kelland et al., 1986 ▸; Lam et al., 1988 ▸; Higgins et al., 1989 ▸; Gale et al., 1981 ▸; Williams et al., 1980 ▸). (d,l)-2-Aminopimelic acid, when incorporated into a peptide, was found to display antibiotic activity against a range of different bacteria and the results suggest that the antibiotic activity was due to interference with the DAP biosynthetic pathway (Berges, DeWolf, Dunn, Grappel et al., 1986 ▸; Berges, DeWolf, Dunn, Newman et al., 1986 ▸). Inhibitors of DHDPS with respect to both ASA and pyruvate produced mixed results (Coulter et al., 1999 ▸; Cox et al., 2000 ▸; Hutton et al., 2003 ▸). Inhibition of Mycobacterium tuberculosis DHDPS by α-ketopimelate and about 35 analogs of α-ketopimelate has been reported by Shrivastava et al. (2016 ▸). They found that α-ketopimelate was the best inhibitor, with an IC50 of about 27 µM. A survey of about 50 2,4-thiazolidinediones and analogous heterocycles for inhibition of E. coli DHDPS found that the best two compounds had IC50 values of about 46 µM (Christoff et al., 2021 ▸). Interestingly, an X-ray crystal structure of one of these compounds with DHDPS from Arabidopsis thaliana indicated that it binds to a pocket adjacent to the allosteric lysine-binding site. A potent inhibitor has been reported that binds to the allosteric lysine binding site. It consists of two linked lysine molecules and has an inhibition constant of about 0.2 mM (Skovpen et al., 2016 ▸). Aceto­pyruvate, a substrate analog of pyruvate, was shown to be an effective slow-binding inhibitor of DHDPS (Karsten et al., 2021 ▸). Presently, no potent broad-spectrum antibacterial compounds against the DAP synthetic pathway have been found and therefore further studies to develop such compounds are crucial.

The three-dimensional structures of DHDPS from E. coli (Blickling et al., 1997 ▸; Dobson, Griffin et al., 2005 ▸; Mirwaldt et al., 1995 ▸), as well as the structures of five point mutants of E. coli DHDPS (Dobson et al., 2004 ▸; Dobson, Devenish et al., 2005 ▸), have been determined. The DHDPS enzyme from E. coli is a tetramer of approximate molar mass 140 000 Da. The subunit mass is estimated to be 28 757 Da based upon the DNA sequence and mass spectrometry (Borthwick et al., 1995 ▸). The enzyme quaternary structure is characterized as a dimer of tight dimers. There are many interactions between the monomers that form the tight dimer, although there are few interactions between the two tight dimers that form the tetramer. The active site is situated in the center of the α/β barrel in each monomer and the active-site Lys161 has access via two entrances from the C-terminal side of the barrel (Dobson et al., 2004 ▸).

The reaction catalyzed by DHDPS is shown in Fig. 1 ▸.

Figure 1.

Figure 1

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The reaction catalyzed by DHDPS. (a) First half-reaction. (b) Second half-reaction.

In the first half-reaction pyruvate binds to the free enzyme, followed by attack of the ɛ-amino group of Lys161 on the carbonyl C atom of the bound pyruvate, leading to the loss of water and the generation of a Schiff-base intermediate (AI). Deprotonation at C3 of the Schiff base intermediate yields an enamine intermediate (AII). The loss of the proton is the step that leads to the ping-pong kinetic mechanism displayed by DHDPS (Karsten, 1997 ▸). After binding of aspartate-β-semialdehyde (ASA) to initiate the second half-reaction, the subsequent steps of the reaction take place, including attack of the enamine on the carbonyl group of ASA leading to the intermediate BI, followed by dehydration to give BII and finally cyclization to generate the product di­hydrodipicolinate. The enzyme is allosterically inhibited by l-lysine (Dobson, Griffin et al., 2005 ▸; Karsten, 1997 ▸). Two molecules of l-lysine bind cooperatively to a site at the dimer interface and, at a saturating concentration, lead to about 90% inhibition of enzyme activity.

2. Materials and methods

The E. coli dapA and dapB genes, which encode DHDPS and dihydrodipicolinate reductase (DHDPR), respectively, were cloned and the resulting His-tagged enzymes were purified according to previously described procedures (Karsten et al., 2018 ▸). The enzyme was expressed in E. coli strain BL21 (DE3) cells from Invitrogen and was manipulated and maintained using standard techniques. The Ni–NTA affinity matrix was purchased from Qiagen. Chemicals were purchased from Sigma (St Louis, Missouri, USA). Isopropyl β-d-1-thio­galactopyranoside (IPTG) was obtained from Gold Biotechnology (St Louis, Missouri, USA). All crystallography solutions were obtained from Hampton Research (Aliso Viejo, California, USA). (S)-2-Bromopropionate was purchased from Sigma.

2.1. Expression and purification

The recombinant plasmid was expressed in E. coli BL21 (DE3) cells cultured on LB/Amp broth and induced with 0.5 mM IPTG. The protein was purified using an Ni–NTA matrix and eluted with buffers ranging between 100 and 300 mM imidazole in lysis buffer at pH 8.0. A Bradford protein assay using bovine serum albumin as the protein standard was performed to confirm the presence and relative abundance of the protein (Bradford, 1976 ▸). Protein peaks with enzymatic activity were separated, pooled, placed into a dialysis bag and dialyzed against 50 mM HEPES buffer pH 7.5 to remove the imidazole. Dialysis was carried out against two 1 l volumes of HEPES buffer at 4°C. SDS–PAGE was used to analyze the purity of DHDPS following the purification process and it was determined to be greater than 95% pure.

2.2. Kinetic studies

DHDPS was assayed as described previously (Karsten, 1997 ▸). The assay is a coupled enzyme assay that contains ASA and pyruvate, the substrates of DHDPS, and the coupling enzyme dihydrodipicolinate reductase that reduces the product of the synthase reaction using NADH. In the enzyme assay, the reductase is present at a tenfold molar excess compared with the synthase and is sufficient to give linear reaction rates and to provide limiting rates by the synthase. The oxidation of NADH is followed at 340 nm using the NADH extinction coefficient of 6220 M −1 cm−1. An initial velocity inhibition pattern was determined in 50 mM HEPES, 100 mM CHES buffer pH 8.0 in the absence and the presence of 6–12 mM concentrations of (S)-2-bromopropionate and varied concentrations of pyruvate (25–250 µM) and a fixed concentration of 0.2 mM for ASA. The kinetics of inactivation of DHDPS by (S)-2-bromopropionate were determined by incubating different concentrations of (S)-2-bromopropionate with 350 µg ml−1 (12 µM) DHDPS in a reaction mixture containing 200 mM HEPES pH 8.0 in a 50 µl final volume at 22°C. To determine the remaining residual activity, 2 µl aliquots of the pre-incubation mixture were transferred to an assay mixture at time zero and at 5 min intervals thereafter. In addition to DHDPS, the assay mixture contained 100 mM CHES, 50 mM HEPES, 2 mM pyruvate, 0.2 mM NADH, 0.2 mM ASA and 259 nM DHDPR in a 1 ml total volume at a pH of 8.0.

2.3. Data analysis

Data were fitted using equations (1) or (2):

2.3.
2.3.

In equation (1), v is the initial rate, V is the maximum rate, A is the substrate concentration, I is the inhibitor concentration, K m is the Michaelis constant and K is is the inhibition constant. In equation (2), Vt is the rate at time t, V 0 is the rate at time zero and k is the decay constant.

2.4. Crystallization

The enzyme–(S)-2-bromopropionate complex was formed in a solution containing 10 mg ml−1 protein and 8 mM (S)-2-bromopropionate. The solution was incubated at room temperature for 10 min. The hanging-drop vapor-diffusion method was used to set up crystal drops consisting of 2 µl protein solution mixed with 2 µl well buffer. Diffraction-quality crystals formed in drops at pH 7.5 in the presence of 4 mM (S)-2-bromopropionate, 20% PEG 3350, 8 mM spermidine (solution No. 38 of the Hampton Research Additive Screen), 0.2 M sodium tartrate and 5.0 mg ml−1 DHDPS. A cryoprotectant solution was prepared by adding glycerol to the crystallization solution to a final concentration of 15%.

2.5. Data collection and processing

X-ray diffraction data were collected on a Dectris PILATUS P200K hybrid pixel detector using Cu Kα radiation from a Rigaku MicroMax 007-HF (Rigaku Americas) rotating-anode generator. Images were processed and scaled using HKL-3000 (Minor et al., 2006 ▸). Data-collection statistics are shown in Table 1 ▸.

Table 1. Data-collection and refinement statistics for the crystal structure of the DHDPS–propionate complex.

Values in parentheses are for the highest resolution shell.

Wavelength (Å) 1.542
Resolution range (Å) 25.17–2.155 (2.232–2.155)
Space group P1
a, b, c (Å) 86.14, 86.333, 107.002
α, β, γ (°) 109.715, 104.244, 99.182
Total reflections 460256
Unique reflections 142598 (13375)
Multiplicity 3.2
Completeness (%) 97.33 (91.45)
Mean I/σ(I) 7.7 (1.2)
Wilson B factor (Å2) 21.79
R merge 0.149 (0.756)
R meas 0.177 (0.989)
R p.i.m. 0.093 (0.599)
CC1/2 0.868 (0.524)
Reflections used in refinement 142542 (13371)
Reflections used for R free 2008 (189)
R work 0.1893 (0.2683)
R free 0.2276 (0.3052)
No. of non-H atoms
 Total 20134
 Macromolecules 17498
 Ligands 158
 Solvent 2478
No. of protein residues 2346
R.m.s.d., bond lengths (Å) 0.008
R.m.s.d., angles (°) 0.93
Ramachandran favored (%) 98.57
Ramachandran allowed (%) 1.43
Ramachandran outliers (%) 0.00
Rotamer outliers (%) 0.11
Clashscore 6.23
Average B factor (Å2)
 Overall 24.08
 Macromolecules 22.96
 Ligands 23.75
 Solvent 32.01
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2.6. Structure solution and refinement

The structure was solved by molecular replacement using Phaser (McCoy et al., 2007 ▸). The structure of dihydro­dipicolinate synthase from E. coli (PDB entry 1yxc; Dobson, Griffin et al., 2005 ▸) was used as the initial model. Eight monomers were found with a Phaser TFZ of 122.6 and LLG of 44698.7. The average r.m.s.d. on Cα atoms for all monomers is 0.13 Å; the all-atom r.m.s.d. is higher by about 0.5 Å. The structure was refined using phenix.refine from the Phenix suite (Liebschner et al., 2019 ▸). Model building and solvent placement were performed using Coot (Emsley et al., 2010 ▸). Validation was performed using MolProbity (Chen et al., 2010 ▸). Refinement statistics are shown in Table 1 ▸.

2.7. Structural alignment and modeling

All structural alignments, modeling and distance measurements were performed using the PyMOL molecular-graphics program (Schrödinger).

3. Results and discussion

3.1. Kinetics of inhibition by (S)-2-bromoproprionate

An inhibition pattern of (S)-2-bromoproprionate versus pyruvate is shown in Fig. 2 ▸. The pattern indicates that (S)-2-bromoproprionate is a competitive inhibitor versus pyruvate. Under these conditions, the K i for (S)-2-bromoproprionate is 8.4 ± 0.7 mM and the K m for pyruvate is 0.23 ± 0.02 mM. The K m for pyruvate is similar to the K m of 0.17 ± 0.03 mM previously reported from a full initial velocity pattern (Karsten, 1997 ▸). (S)-2-Bromoproprionate does not bind to the enzyme with high affinity, as indicated by a dissociation constant that is almost 40 times greater than the K m for pyruvate. Although (S)-2-bromoproprionate shares some structural similarities with pyruvate, the differences are significant. The substitution of bromine in place of the carbonyl O atom is one. The most important difference is probably the difference in geometry at C2 between pyruvate and (S)-2-bromoproprionate. The tetrahedral configuration at C2 of (S)-2-bromoproprionate most likely does not allow the usual interactions within the active site that favor pyruvate binding.

Figure 2.

Figure 2

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Inhibition pattern of (S)-2-bromoproprionate versus pyruvate. The (S)-2-bromoproprionate concentrations were 0 mM (squares), 6 mM (circles) and 12 mM (triangles). Pyruvate was varied between 0.03 and 0.25 mM. The other assay components were fixed at 0.2 mM ASA, 100 mM CHES, 50 mM HEPES, 0.2 mM NADH, 250 nM DHDPR and 20 nM DHDPS. The assays were carried out at pH 8.0. The data points are the experimentally determined values and the lines are derived from a fit of the data using equation (1).

(S)-2-Bromoproprionate contains a good leaving group, making the molecule susceptible to nucleophilic attack at C2. This characteristic of the inhibitor raises the possibility that attack of Lys161 on C2 of (S)-2-bromoproprionate could lead to covalent modification of the enzyme at Lys161 and thus to inactivation of the enzyme. This possibility was investigated by pre-incubating the enzyme with (S)-2-bromoproprionate and checking the residual enzyme activity over time. The results of the experiment are shown in Fig. 3 ▸. The figure shows a loss of DHDPS activity over time that is dependent upon the concentration of (S)-2-bromoproprionate. The rate constants for inactivation at 2, 4 and 8 mM (S)-2-bromoproprionate are 0.026 ± 0.001, 0.051 ± 0.002 and 0.095 ± 0.003 min−1, respectively, based upon a fit of the data at each concentration of (S)-2-bromoproprionate using equation (2). Also shown in Fig. 3 ▸ is an experiment in which the enzyme was pre-incubated with 8 mM (S)-2-bromoproprionate and 2 mM pyruvate. Under these conditions pyruvate provides complete protection from inactivation and is consistent with the results from the inhibition pattern shown in Fig. 2 ▸, indicating that (S)-2-bromoproprionate is a competitive inhibitor versus pyruvate.

Figure 3.

Figure 3

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DHDPS was pre-incubated in the absence (filled circles) or the presence of 2 mM (circles), 4 mM (filled squares) or 8 mM (squares) (S)-2-bromoproprionate. The enzyme was also pre-incubated with both 2 mM pyruvate and 8 mM (S)-2-bromoproprionate (filled diamonds). Vt is the rate at time t and V 0 is the rate at time zero. In order to determine the residual activity of DHDPS at the time intervals indicated, 2 µl of the pre-incubation mixture was transferred to an enzyme assay containing final concentrations of 100 mM CHES, 50 mM HEPES. 2 mM pyruvate, 0.2 mM ASA, 0.2 mM NADH, 259 nM DHDPR and 24 nM DHDPS at pH 8.0. The lines are derived from a fit of the data using equation (2).

3.2. Structure of DHDPS covalently modified by (S)-2-bromoproprionate

The structure of E. coli DHDPS deposited in the PDB (PDB entry 6nva) shows two tetramers containing a total of eight monomers (labeled A–D and E–H), each of which contains one active site. In seven of the eight monomers there is a glycerol molecule bound within a cavity that extends from the lysine allosteric site towards the active site. Every active site has Lys161 modified by (S)-2-bromoproprionate to form a covalent lysine–propionate adduct. The position of the adduct and its interactions with active-site groups is similar from one active site to the other. The largest variability in the position of the propionate within the active site involves the distance between one α-carboxyl O atom of propionate and the hydroxyl group of Tyr133. This distance varies from 2.6 to 3.8 Å. The average distance calculated using all monomers is 3.43 Å. There are some minor compensatory changes in distance between active-site groups and propionate that are associated with the different Tyr133 to carboxyl group distances. For the figures shown below subunit B was used as representative of the enzyme–propionate structure since the Tyr133 to carboxyl group distance is 3.5 Å in this subunit, which is close to the average distance for all subunits.

The covalent modification of DHDPS by (S)-2-bromo­proprionate is corroborated by the X-ray crystal structure of the enzyme solved in the presence of (S)-2-bromoproprionate. Fig. 4 ▸ shows the structure of the active site with the bound adduct formed from (S)-2-bromoproprionate (Fig. 4 ▸ a) and, for comparison, the active site with the bound adduct formed from reaction of the enzyme with the natural substrate pyruvate (Fig. 4 ▸ b). The figure also shows the distances between the bound adduct and the active-site groups that are in close proximity (within 3.5 Å). The ɛ-amino group of Lys161 interacts with pyruvate by attack on the carbonyl C atom of pyruvate with loss of water and generation of a Schiff-base intermediate. The α-carboxyl group forms hydrogen-bonding interactions with the hydroxyl side chain of Thr45 and the backbone N atoms of Thr45 and Thr44. These interactions place the carboxyl group in essentially the same plane as the α-methyl group and the imine bond involving the N atom of Lys161.

Figure 4.

Figure 4

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(a) The covalent adduct formed on reaction of DHDPS with pyruvate in the active site of the enzyme (PDB entry 3du0; Devenish et al., 2008 ▸). (b) The active site of the enzyme with the covalent adduct formed from reaction with (S)-2-bromoproprionate (PDB entry 6nva). The hydrogen-bonding interactions are shown for each structure. This figure was generated using PyMOL (Schrödinger).

Compared with pyruvate, there are a number of differences in how the covalently bound propionate formed from reaction with (S)-2-bromoproprionate interacts with active-site groups. The reaction between the ɛ-amino group of Lys161 and C2 of (S)-2-bromoproprionate will lead to elimination of Br− and the generation of a single bond between propionate and Lys161, in contrast to the imine bond formed with pyruvate. C2 of the bound propionate is chiral and in the R-configuration. Since the 2-bromopropionate used to modify the enzyme was in the S-configuration, this indicates that there was back-side attack of the ɛ-amine of Lys161, leading to inversion of configuration, in an SN2-type reaction.

In the structure formed with pyruvate, one of the O atoms of the α-carboxyl group interacts with the backbone N atoms of Thr44 (3.4 Å) and Thr45 (2.9 Å) and also with the hydroxyl side chain of Thr45 (2.8 Å). As seen in the (S)-2-bromoproprionate structure, one α-carboxyl O atom interacts in a similar manner with Thr44 and Thr45. The second O atom of the α-carboxyl group in the pyruvate structure is 2.8 Å from the backbone N atom of Thr44 and 3.5 Å from the side-chain hydroxyl group of Thr44. In contrast, the second carboxyl O atom in the (S)-2-bromoproprionate structure is 3.3 Å from the backbone N atom of Thr44 and more than 3.5 Å from the hydroxyl group of Thr44. In addition, this O atom is 3.5 Å from the hydroxyl group of Tyr133. In both the pyruvate structure and the (S)-2-bromoproprionate structure Tyr133 is within 3.4 Å of the Nɛ atom of Lys161. In general, the interactions between active-site groups and the covalently bound adducts in the pyruvate and (S)-2-bromoproprionate structures are pretty similar.

The carboxyl group of the adduct formed from (S)-2-bromoproprionate is in a similar location in the active site of DHDPS as the carboxyl group of pyruvate, suggesting that the carboxyl group of (S)-2-bromoproprionate is involved in the binding and orientation of (S)-2-bromoproprionate, helping to place it in position for attack by Lys161. The differences between the structures formed from pyruvate and (S)-2-bromoproprionate can best be observed in the overlay of the two structures shown in Fig. 5 ▸. The α-methyl groups of the two structures are in a similar position, but the carboxyl group of the covalently bound propionate is displaced somewhat towards Tyr133 compared with the pyruvate structure. The most significant difference in the two structures involves Lys161. There is a significant twisting of the aliphatic chain of Lys161 in the structure formed from (S)-2-bromoproprionate and this movement displaces the position of the Nɛ atom of Lys161 and allows the rest of the bound propionate to overlay fairly closely with the covalently bound pyruvate structure. Whether this twisting movement of Lys161 occurs on the initial binding of (S)-2-bromoproprionate or as a consequence of the reaction of Lys161 with (S)-2-bromoproprionate cannot be determined from the present data.

Figure 5.

Figure 5

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The figure shows an overlay of the active site of the enzyme with the covalent adducts formed with either pyruvate (blue) or (S)-2-bromoproprionate (green). This figure was generated using PyMOL (Schrödinger).

4. Conclusion

The inhibitor (S)-2-bromoproprionate binds to DHDPS with low affinity and consequently is not a likely candidate as a lead compound for inhibitor development. However, the results do suggest that a compound with a good leaving group at the appropriate position in a higher affinity inhibitor should lead to inactivation of the enzyme. A tight-binding inhibitor with an appropriate reactive group could be a possible drug candidate.

Supplementary Material

PDB reference: Escherichia coli dihydro­dipicolinate synthase, covalent adduct with (S)-2-bromopropionate, 6nva

Acknowledgments

The authors wish to thank Fariha Sultana for her technical assistance in this project and Dr Paul Sims at the University of Oklahoma for the use of some of his instrumentation to complete these studies.

Funding Statement

This work was funded by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM1034472. This work reports data obtained at the University of Oklahoma Macromolecular Crystallography Laboratory, which is supported, in part, by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (Grant P20GM103640).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PDB reference: Escherichia coli dihydro­dipicolinate synthase, covalent adduct with (S)-2-bromopropionate, 6nva

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