Elsevier

Bioorganic Chemistry

Volume 57, December 2014, Pages 242-250
Bioorganic Chemistry

Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase – The importance of accommodating the active site water

https://doi.org/10.1016/j.bioorg.2014.08.003Get rights and content

Highlights

  • Competitive inhibition of N. meningitidis DAH7P synthase by substrate mimics.

  • Co-accommodation of active site water and inhibitor is important for inhibition.

  • Key catalytic residues of DAH7P synthase identified from inhibitor bound structures.

Abstract

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9 ± 0.4 μM. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated.

Introduction

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which is responsible for the biosynthesis of the aromatic amino acids and other important aromatic metabolites in plants and microorganisms [1], [2]. In mammals these essential metabolites are obtained from dietary sources, and the enzymes of the shikimate pathway are absent. Therefore, the enzymes of this pathway have been identified as promising targets for the development of new antibacterial therapeutics [3], [4], [5].

DAH7PS catalyses the condensation between d-erythrose 4-phosphate (E4P, 2) and phosphoenol-pyruvate (PEP, 1) to generate DAH7P (3, Fig. 1). Many of the key mechanistic details of the reaction have been elucidated through various labelling, structural and alternative-substrate studies [3], [6], [7], [8], [9], [10], [11], [12]. The ordered sequential reaction proceeds stereospecifically with respect to both substrates, with the si face of PEP attacking the re face of E4P. The aldol-like reaction takes place with cleavage of the C–O bond of the phosphate group of PEP, requiring water to act as a co-substrate. The initial nucleophilic attack of PEP on E4P is predicted to be promoted by coordination of the E4P aldehydic oxygen to the essential divalent metal ion, giving an oxocarbenium ion intermediate 4 (Fig. 1) [3], [6]. The addition of an active site nucleophilic water results in a phosphohemiketal tetrahedral intermediate 5, which forms acyclic DAH7P on the elimination of phosphate. The stereochemistry of the tetrahedral intermediate 5, which is lost on the elimination of phosphate, is determined by the face of the oxocarbenium ion 4 that is attacked by the nucleophilic water. Therefore, the ability of the active site to accommodate and activate a water molecule is an important aspect of DAH7PS catalysis and, furthermore, may have implications for inhibitor design [3], [13].

DAH7PS enzymes have been structurally and functionally characterised from a variety of sources [6], [13], [14], [15], [16], [17], [18], [19]. While all known enzymes have similar active site architectures supporting a common catalytic mechanism and share a common (β/α)8-barrel core, there is considerable sequence divergence between enzymes from different sources leading to the classification of DAH7PS enzymes into two types [20]. Type I enzymes are further divided into type Iα and type Iβ subfamilies, which differ in sequence and their allosteric regulatory machinery. The only structurally characterised type II enzyme is from Mycobacterium tuberculosis [16], [21].

Active site inhibitors have been previously reported for the phenylalanine-sensitive type Iα DAH7PS from Escherichia coli (EcoDAH7PS) and the type II enzyme from M. tuberculosis (MtuDAH7PS) [3], [12], [22], [23], [24]. PEP-like analogues were found to inhibit EcoDAH7PS, with the most potent inhibitor being the vinyl phosphonate, (E)-2-methyl-3-phosphonoacrylate 6, followed by the (R)-phospholactate 7 [23]. (S)-Phospholactate 8 was the poorest inhibitor. The rationale for a ten-fold difference in potency of the two phospholactate stereoisomers is not clear, as no structures of the inhibited enzymes were determined, but may relate to the stereochemistry of the transient phosphohemiketal tetrahedral intermediate 5. The vinyl phosphonate 6 was found to have a Ki of 4.7 ± 0.7 μM, which approaches the Km for PEP of 2 μM for this enzyme [23]. Inhibition studies on the type II MtuDAH7PS were carried out using extended (R)- and (S)-phospholactates of variable chain length bearing an additional phosphate moiety in order to better mimic the key functionality of the tetrahedral intermediate (bisphosphates 9 and 10) [3], [24]. In each case the compounds bearing (R)-configuration were found to inhibit enzymatic activity more potently than the respective (S)-enantiomers. The seven-carbon containing (R)-bisphosphate 9 had a Ki value of 0.36 ± 0.05 μM, consistent with this compound significantly out-competing the natural substrate PEP (KmPEP of 37 μM) [3], [24].

The genome of Neisseria meningitidis, the causative agent of pyogenic meningitis and meningococcal septicaemia, encodes a single type Iα DAH7PS (NmeDAH7PS) [17], [25]. The crystal structure of NmeDAH7PS, which was recently determined in our laboratory, revealed that the active site of NmeDAH7PS shares many similarities to the DAH7PS from E. coli [33]. The active site Mn2+ is coordinated in a trigonal–bipyramidal fashion by four conserved protein residues, Cys63, His270, Asp304 and Asp324 [17], leaving one coordination site available for the aldehyde moiety of E4P. PEP binding occurs through interaction with the highly conserved residues Arg167, Arg236, Lys188, Arg94 and Lys99. The predicted E4P binding site consists of a highly conserved motif involving the residues Lys99, Pro100, Arg101 and Thr102.

Here we examine the inhibition of NmeDAH7PS by the active site directed PEP-mimicking inhibitors 6, 7 and 8 in order to assess their efficacies against a DAH7PS enzyme from an important pathogen. Furthermore, we report for the first time crystal structures of a DAH7PS, here NmeDAH7PS, in complex with each of these inhibitors, revealing that these inhibitors do accurately mimic the binding modes predicted for the oxocarbenium ion 4 and tetrahedral intermediates 5, along with the co-coordination of a water molecule. Our findings allow refinement of the DAH7PS mechanism, aid the identification of the nucleophilic water in this type Iα enzyme and guide further structure-based inhibitor design of this enzyme.

Section snippets

Inhibitor synthesis

(R)-Phospholactate 7, (S)-phospholactate 8 and the vinyl phosphonate 6 were synthesised according to the previously reported methods [23]. Concentration determination of the vinyl phosphonate 6 was achieved by comparison of signal integrals from 31P NMR spectroscopy using 85% phosphoric acid as a standard at sufficiently long relaxation times. Phospholactate concentrations were determined via Lanzetta assay for phosphate detection as previously described [3], [26].

Inhibition assays

Enzyme activity was measured

NmeDAH7PS is inhibited by PEP analogues

Previous studies have shown that both EcoDAH7PS and MtuDAH7PS were inhibited by compounds that possess either a planar trigonal or a tetrahedral geometry at C2 [3], [22], [23]. Both phospholactate stereoisomers 7 and 8 and the vinyl phosphonate 6 were synthesised and tested as inhibitors of NmeDAH7PS. All three compounds were found to inhibit NmeDAH7PS competitively with respect to substrate PEP. The most potent inhibitor was the vinyl phosphonate 6, with an observed Ki value of 3.9 ± 0.4 μM. This

Conclusion

Three PEP mimics were found to be inhibitors of NmeDAH7PS and to occupy the PEP binding pocket of the active site of this enzyme. Analysis of the first co-crystal structures of these small but highly atom-efficient inhibitors with their target enzyme highlight the importance of the accommodation of the nucleophilic water molecule in the active site for potent inhibition. From our studies we conclude that planar rather than tetrahedral PEP-mimics are more promising candidates for inhibition of

Acknowledgments

This research was undertaken on the MX1 and MX2 beamlines at the Australian Synchrotron, Victoria, Australia. Funding is gratefully acknowledged for these studies from the New Zealand Marsden Fund (UOC1105). S.R. is grateful for financial support from a University of Canterbury Doctoral Scholarship and a New Zealand International Doctoral Research Scholarship.

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    Current address: Technische Universität Berlin, Fakultät II Mathematik und Naturwissenschaften, Institut für Chemie / OC / Biologische Chemie, Müller-Breslau-Straße 10, L3, 10623 Berlin, Germany.

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