Novel inhibitors of dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of L-lysine and meso-diaminopimelate biosynthesis, which is the condensation of (S)-aspartate-β-semialdehyde (ASA) and pyruvate into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS has been an attractive antibiotic target because L-lysine and meso-diaminopimelate are cross-linking components between peptidoglycan heteropolysaccharide chains in bacterial cell walls. Studies revealed that mutant auxotrophs for diaminopimelate undergo lysis in the absence of diaminopimelate in the medium; therefore the assumption is that strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and L-lysine biosynthesis in bacteria and would stop or decrease bacterial growth (eventually leading to bacterial death). In this work, the DHDPS inhibitor design is focused on the allosteric site of the enzyme. It was proposed that a compound mimicking binding of two L-lysine molecules at the allosteric site at the enzyme’s dimer-dimer interface would be a more potent inhibitor than the natural allosteric inhibitor of this enzyme, L-lysine. This inhibitor (R,R-bislysine) was synthesized as a racemic mixture, which was then separated with the aid of chiral HPLC. The mechanism of feedback inhibition of DHDPS from Campylobacter jejuni with its natural allosteric modulator, L-lysine, and its synthetic mimic, R,R-bislysine, is studied in detail. It is found that L-lysine is a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. R,R-bislysine is a mixed partial inhibitor with respect to pyruvate and a noncompetitive partial inhibitor with respect to ASA, with an inhibition constant of 200 nM. Kinetic evaluation of each DHDPS mutants (Y110F, H56A, H56N, H59A and H59N) has revealed amino acids responsible for the inhibitory effect of L-lysine, R,R-bislysine, and we have found that R,R-bislysine is a strong submicromolar inhibitor of Y110F, H56A, H56N and H59N

Structure-based mechanism of hydratase-aldolases in the Type I aldolase superfamily and structures of tautomerase superfamily members

Polycyclic Aromatic Hydrocarbons (PAHs) are composed of multiple benzene-like rings and their bacterial catabolism has potential utility for bioremediation. In the breakdown of each ring, oxygenation is followed by ring cleavage and side chain removal, the latter of which is catalyzed by hydratase-aldolases. These enzymes belong to the N-acetylneuraminate lyase (NAL) subgroup of the Type I aldolase superfamily. NahE and PhdJ are hydratase-aldolases that process benzylidenepyruvate compounds in PAH degradative pathways from Pseudomonas putida G7 and Mycobacterium vanbaalenii PYR-1, respectively. Crystal structures of these enzymes in liganded and unliganded states are reported here and reveal new details about their catalytic mechanisms. Stabilization of the developing hydroxide anion during hydration could involve the amide nitrogen of Asn-157 (NahE numbering), conserved in hydratase-aldolases, but not other NAL subgroup members. For NahE, Tyr-155 might act as the general base for addition, but the identity of the base is less certain for PhdJ. The crystal structure of PhdG reveals a carbinolamine intermediate hydrogen bonding to Tyr-152, consistent with the participation of the residue in Schiff base formation. In addition, this dissertation describes structures within the tautomerase superfamily (TSF). The enzymes reported are from 4-oxalocrotonate tautomerase (4-OT), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD) subgroups. 4-OT catalyzes the ketonization of 2-hydroxymuconate (2-HM) in the meta-fission pathway. cis-CaaD and MSAD respectively catalyze dehalogenation and decarboxylation reactions in the catabolic 1,3-dichloropropene pathway, respectively. TSF members have a catalytic Pro-1 and share a common β-α-β fold. TSF members designated Ps01740, Pt0534, and Fused 4-OT have sequence similarity with the cis-CaaD and 4-OT subgroups and may provide insight into gene duplication and fusion events in the TSF. These enzymes have tautomerase activity and low or absent cis-CaaD activity. Liganded and unliganded structures suggest a structural basis for their activation. The trimeric structure of Fused 4-OT is unusual in that one monomer is inverted relative to the others. Mechanisms for 2-HM tautomerization are proposed based on a 2-HM-bound structure and kinetic parameters of mutants. Crystal structures for the MSAD homologues BP4401 and YusQ were obtained, and provide clues about their activitiesBiochemistr

Identification of the evolutionary divergence in DHDPS and DHDPR

DHDPS and DHDPR are the first two committed steps in the DAP pathway: a pathway responsible for the biosynthesis of lysine. It is only present in bacteria and plants making an important biological target. While DHDPS exists in a homotetrameric “dimer of dimers” formation in both bacteria and plants, the arrangement of monomers is different. In bacteria, the dimers face toward each other in a front to front arrangement. However, in plants, the orientation of the dimers is flipped into a back to back arrangement. An evolutionary difference is also observed in DHDPR. In bacteria, the protein exists in a homotetrameric conformation whereas in plants it has been shown to exist in a dimeric conformation. The exact reason for these differences in structure remain unclear but it is thought to due to evolutionary changes between the two organism types. In this study, a lycophyte DHDPS from Selaginella moellendorffii was found to exist in a substrate mediated equilibrium between dimer and tetramer, with no ligands bound. When the substrate pyruvate is bound to the enzyme, the equilibrium shifted to the tetrameric species. However, in the presence of the allosteric inhibitor lysine, the equilibrium was found to shift to a dimeric species in solution. This equilibrium could exist as a “missing link” in the evolution of the plant type quaternary structure of the DHDPS enzyme. Another subject of investigation was the characterisation of red, green and brown algal DHDPRs. The quaternary state of these species was found to be dimeric in nature. This corresponds to the proposed evolutionary lineage in which most of these species exist after the plant type species in the lineage. The exception to this is the green alga Chlamydomonas reinhardtii DHDPR which exists in an equilibrium between tetramer and dimer. As this organism lies in the evolutionary lineage between bacterial and plant forms, it is possible that this organisms DHDPR exists as the “divergence point” between these two species. C. reinhardtii DHDPR also contains a disulfide-dependent dimer interface. In the presence of reducing agent, the enzyme exists in an exclusively dimeric state. These evolutionary lineages could be applied to other enzyme evolution systems from the DAP pathway and beyond

Determination of the Structural Allosteric Inhibitory Mechanism of Dihydrodipicolinate Synthase

Dihydrodipicolinate Synthase (EC 4.3.3.7; DHDPS), the product of the dapA gene, is an enzyme that catalyzes the condensation of pyruvate and S-aspartate-β-semialdehyde (ASA) into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS catalyzes the first committed step in the biosynthesis of ʟ-lysine and meso-diaminopimelate; each of which is a necessary cross-linking component between peptidoglycan heteropolysacharide chains of bacterial cell walls. Therefore, strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and ʟ-lysine biosynthesis in bacteria leading to decreased bacterial growth and cell lysis. Much attention has been given to targeting the active site for inhibition; however DHDPS is subject to natural feedback inhibition by ʟ-lysine at an allosteric site. In DHDPS from Campylobacter jejuni ʟ-lysine is known to act as a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. Little is known about how the protein structure facilitates the natural inhibition mechanism and mode of allosteric signal transduction. This work presents ten high resolution crystal structures of Cj-DHDPS and the mutant Y110F-DHDPS with various substrates and inhibitors, including the first reported structure of DHDPS with ASA bound to the active site. As a body of work these structures reveal residues and conformational changes which contribute to the inhibition of the enzyme. Understanding these structure function relationships will be valuable for the design of future antibiotic lead compounds. When an inhibitor binds to the allosteric site there is meaningful shrinkage in the solvent accessible volume between 33% and 49% proportional to the strength of inhibition. Meanwhile at the active site the solvent accessible volume increases between 5% and 35% proportional to the strength of inhibition. Furthermore, inhibitor binding at the allosteric site consistently alters the distance between hydroxyls of the catalytic triad (Y137-T47-Y111') which is likely to affect local pKa's. Changes in active site volume and modification of the catalytic triad would inhibit the enzyme during the binding and condensation of ASA. The residues H56, E88, R60 form a network of hydrogen bonds to close the allosteric site around the inhibitor and act as a lid. Comparison of ʟ-lysine and bislysine bound to wt-DHDPS and Y110F-DHDPS indicates that enhanced inhibition of bislysine is most likely due to increased binding strength rather than altering the mechanism of inhibition. When ASA binds to the active site the network of hydrogen bonds among H56, E88 and R60 is disrupted and the solvent accessible volume of the allosteric site expands by 46%. This observation provides some explanation for the reduced affinity of ʟ-lysine in high ASA concentrations. ʟ-Lysine, but not other inhibitors, is found to induce dynamic domain movements in the wt-DHDPS. These domain movements do not appear to be essential to the inhibition of the enzyme but may play a role in cooperativity between monomers or governing protein dynamics. The moving domain connects the allosteric site to the dimer-dimer interface. Several residues at the weak dimer interface have been identified as potentially involved in dimer-dimer communication including: I172, D173, V176, I194, Y196, S200, N201, K234, D238, Y241, N242 and K245. These residues are not among any previously identified as important for formation of the quaternary structure

Lysine biosynthesis: synthesis of enzyme inhibitors and substrates

Two distinct biosynthetic pathways to the essential amino acid L-lysine (A) are found in nature. The -aminoadipate pathway operates in fungi and yeasts. The diaminopimelate (DAP) pathway occurs in bacteria and higher plants. Our studies were concerned with the DAP pathway and particularly with the first two steps of this pathway. These steps involve condensation of L-aspartate--semialdehyde (ASA) (B) with pyruvate (C) to form L-2,3-dihydrodipicolinic acid (DHDPS) (D) and subsequent reduction to L-2,3,4,5-tetrahydrodipicolinic acid (THDPA) (E). The first step is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS). The second step is catalysed by the enzyme dihydrodipicolinate reductase (DHDPR) and utilises NADPH as a co-factor. (Fig 6269A) Our primary objective was the inhibition of this biosynthetic pathway. Inhibitors of this pathway have potential as antibacterial or herbicidal agents. A number of substrate analogues of the DHDPS and DHDPR enzymes were prepared and tested as inhibitors of these enzymes. In previous studies by our group, heterocyclic compound (F) showed promising activity. In our work, a number of analogues of compound (F) were prepared. Inhibition studies with these compounds constituted a valuable insight into the characteristics of these enzymes. The level of inhibition with these compounds and for a range of other substrate analogues indicate high substrate selectivity for the enzymes. (Fig 6269B) The condensation catalysed by DHDPS is mechanistically interesting. In chemical terms, C-C bond formation is commonly a high energy process involving highly reactive compounds such as organometallic agents or strong bases. In such cases the strategy must be directed towards protecting other functionality or introducing it at a later stage. Clearly, achieving this task under mild conditions with the high regio- and stereoselectivity associated with enzymic catalysis could be of tremendous advantage. Once again, however, we were restricted by the high substrate specificity of the DHDPS enzyme. In earlier studies by our group, Dr J.E. McKendrick found evidence of substrate activity for 2- and 3-methyl substituted ASA, (G) and (H), respectively. In our work, an improved preparation of these compounds was developed and the subsequent biotransformations with DHDPS were examined both qualitatively and quantitatively. Interestingly, compound (G) was shown to display a greater substrate activity than ASA. The preparation of ASA methyl ester (I) was achieved. This compound also displayed a moderate level of substrate activity and was the only compound found to show substrate activity for a DHDPS/DHDPR coupled substrate assay. (Fig 6269C) A significant proportion of our effort was concentrated on the investigation of glutamate--semialdehyde (GSA) (J), the higher homologue of ASA, as a substrate of DHDPS. Problems were encountered with earlier studies in this area because of cyclisation of GSA, even in protected forms. To counter this problem two novel strategies were considered. The first involved in situ enzymic deprotection of the N-acetyl derivative of GSA. Although this was successful for N-Acetyl-ASA, problems with cyclisation were once again experienced for the GSA equivalent. The second strategy utilises the reversible nature of enzymic catalysis. The synthesis of the proposed 7-membered heterocyclic product (K) of DHDPS catalysed condensation of GSA and pyruvate is not a trivial task. Some progress was made and further direction detailed. Further validation for this study was demonstrated on preparation of 5-hydroxyproline (L). Compound (L) is a cyclic equivalent of GSA and was found to display clear evidence of substrate activity. (Fig 6269D

Design and Synthesis of Potential Novel Antibiotic Compounds Utilising Photoredox Catalysis

The continued emergence of widespread antibiotic resistance over the prior several decades poses an increasingly severe worldwide challenge to public health. Several frontline antibiotic treatments are being rendered obsolete due to the advent of numerous bacterial resistance mechanisms, an issue further compounded by the lack of antibiotics currently residing within the antibacterial drug discovery pipeline that operate via previously unexploited mechanisms of action. There are numerous underlying issues that have propagated this unsavoury situation, some specific to antibiotic drug development and others that negatively impact the field of drug discovery as a whole. One of the latter issues centres around the implementation of high throughput target-based screening of suboptimal compound libraries for hit identification, and the narrow range of synthetic methodologies used to explore chemical space within such compound collections. Dihydrodipicolinate synthase (DHDPS) constitutes a promising biomolecular target for novel antibiotic therapies due to its key role in the biosynthesis of essential amino acid L-lysine, a process widely specific to bacteria. Despite several prior campaigns and the development of micromolar potency inhibitors of DHDPS through target–based screening approaches, so far no compounds have been developed that display in vitro antibacterial activity in the subsequent phenotypic screens. In silico screening constitutes an invaluable range of techniques used in the identification of potential hit compounds that has been implemented to great effect in numerous drug discovery campaigns, including the discovery of novel antibacterial compounds, often aiding in the design of more focused compound libraries for assessment in vitro. Photoredox catalysis has emerged as a powerful synthetic tool for enabling access to previously unexplored regions of chemical space especially within medicinal chemistry contexts, facilitating highly chemoselective activation of reagents under benign reaction conditions. Sulfonylhydrazones are well established reagents within the field of organic synthesis capable of undergoing a myriad of transformations. Recent reports concerning the photocatalytic activation of hydrazone substrates to enable radical cyclisations served as the basis for the initial interest in developing related methodologies to generate desired compounds in the search for novel antibacterial agents. In this thesis is described the design and synthesis of potential novel antibacterial compounds, initially utilising pharmacophore searches and qualitative in silico docking investigations to identify molecular scaffolds of interest as synthetic targets. The development of a novel photoredox reaction for the generation of sulfone hit structures from sulfonyl hydrazone starting materials is described, including exploration of the substrate scope and reaction mechanism studies. The synthesis of additional in silico derived hit structures is also described, as well as attempts made to expand the synthetic utility of the developed photocatalytic methodology. Initial evaluation of antibacterial activity of the compound collection is described including preliminary discussion of structure activity relationships as a foundation for the derivation of future work. The final chapter contains technical experimental details and characterisation data pertaining to the previously discussed work

Synthesis and Testing of Inhibitors of Dihydrodipicolinate Synthase

There are two distinct biosynthetic pathways to the essential amino acid L-lysine (A). The diaminopimelate pathway to L-lysine occurs in higher plants and bacteria. The second pathway known is the alpha-aminoadipate pathway and is found to operate in fungi and yeasts. This thesis will deal with only the diaminopimelate pathway to L-lysine and in particular with the first step, which involves the condensation of L-aspartic acid beta-semialdehyde (ASA) (B) with pyruvate (C) to form L-dihydrodipicolinate (DHDP) (D). The mechanism of formation of L-DHDP (D) was studied using electrospray mass spectrometry. The synthesis and testing of potential inhibitors of dihydrodipicolinate synthase (DHDPS) was also studied. [diagram] L-ASA is a substrate of the first enzyme of the diaminopimelate pathway to L-lysine. A former co-worker in the group, Dr D. Tudor had developed a route to 1-ASA as the trifluoroacetate salt. This route was low yielding, approximately 14% for four steps, thus a higher yielding route was developed utilising the para-methoxybenzyl (PMB) ester protecting group. This material was not suitable for use in the biochemical assay as an impurity from the deprotection stage was found to absorb strongly at the wavelength used for the our enzyme assay system. An improved procedure for the synthesis of L-ASA was developed increasing the overall yield of the procedure to approximately 48% for the same four steps. The synthesis of L-ASA as its trifluoroacetate salt allowed a number of analogues of 1-ASA to be prepared with some synthetic modification of the original route. The compounds prepared this way were alkylated derivatives of L-ASA. The alpha-methyl ASA (E) was prepared by the reaction of methyl iodide with the anion generated from treating diprotected allylglycine with lithium diisopropylamide (LDA). It proved to be a poor inhibitor but initial studies suggest that it may be a reasonably good substrate for DHDPS. A number of other derivatives were prepared including beta-methyl ASA (F) which again was a poor inhibitor but was found to be a good substrate for DHDPS (beta-methyl ASA is utilised at approximately 20% of the rate that L-ASA is consumed). A number of other derivatives and analogues of L-ASA were prepared. A number of heterocyclic compounds were prepared as analogues of DHDP and were tested for inhibitory effects with DHDPS. These compounds were prepared by a 1,3-dipolar cycloaddition of a nitrile oxide onto an alkene or alkyne. The isoxazolines produced had a general structure (G). These compounds were found to be poor inhibitors of DHDPS with none showing inhibition below 1 mM. The ring opened isoxazolines (H) were prepared as analogues of pyruvate but again they proved to be poor inhibitors of DHDPS. [diagram] An attempt to synthesise glutamic acid gamma-semialdehyde (I) starting from glutamic acid was undertaken. The compound isolated from the series of reactions was found to have cyclised and was stable as the carbinolamine (J). This route was abandoned due to the cyclisation of the product. [diagram] A number of pyridinedicarboxylic acid derivatives and analogues were prepared to test for inhibitory activity. The pyridine-2,6-dicarboxylic acid N-oxide (K) and the pyridine-2,6-dinitrile (L) showed very good inhibitory activity. These compounds were studied in detail to determine the type of inhibition they showed. The two compounds (K) and (L) were found to be non-competitive inhibitors of DHDPS. A number of other saturated and unsaturated analogues of L-DHDP were prepared and tested for inhibitory action. A study of the mechanism of DHDPS was undertaken using electrospray mass spectrometry to detect enzyme bound intermediates. The electrospray mass spectrometer was able to provide evidence for a number of pyruvate analogues bound to the enzyme as Schiff's bases. No evidence for L-ASA bound to DHDPS could be found. Further to these studies was the need to preserve stocks of DHDPS used for inhibitor testing and biotransformations. A study of the immobilisation of DHDPS on Eupergit resins was undertaken to determine the feasibility of this technique as a method of obtaining reusable DHDPS. The studies found that only up to 18% of the initial sample of DHDPS was bound to the beads. This suggests that this may not be a suitable method for the immobilisation of DHDPS, however DHDPS bound to the beads was found to have long term stability

Plant primary metabolism regulated by nitrogen contributes to plant-pathogen interactions.

Nitrogen contributes to plant defense responses by the regulation of plant primary metabolism during plant–pathogen interactions. Based on biochemical, physiological, bioinformatic and transcriptome approaches, we investigated how different nitrogen forms (ammonium vs. nitrate) regulate the physiological response of cucumber (Cucumis sativus) to Fusarium oxysporum f. sp. cucumerinum (FOC) infection. The metabolic profile revealed that nitrate-grown plants accumulated more organic acids, while ammonium-grown plants accumulated more amino acids; FOC infection significantly increased levels of both amino acids and organic acids in the roots of ammonium-grown plants. Transcriptome analysis showed that genes related to carbon metabolism were mostly up-regulated in plants grown with nitrate, whereas in ammonium-grown plants the up-regulated genes were mostly those that were related to primary nitrogen metabolism. Root FOC colonization and disease incidence were positively correlated with levels of root amino acids and negatively correlated with levels of root organic acids. In conclusion, organic acid metabolism and expression of related genes increased under nitrate, whereas ammonium increased the level of amino acids and expression of related genes; these altered levels of organic acids and amino acids resulted in different tolerances to FOC infection depending on the nitrogen forms supplied