Elsevier

Biochimie

Volume 86, Issues 4–5, April–May 2004, Pages 311-315
Biochimie

Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate

https://doi.org/10.1016/j.biochi.2004.03.008Get rights and content

Abstract

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) mediates the first unique reaction of (S)-lysine biosynthesis in plants and microbes—the condensation of (S)-aspartate-β-semialdehyde ((S)-ASA) and pyruvate. It has been shown that DHDPS is partially feedback inhibited by (S)-lysine; it is suggested that this mechanism regulates flux through the DAP biosynthetic pathway. Others have characterised DHDPS from Escherichia coli with respect to (S)-lysine inhibition. They have concluded that, with respect to pyruvate, the first substrate of the reaction, DHDPS shows uncompetitive inhibition: as such, they further suggest that (S)-lysine inhibits DHDPS via interaction with the binding site for the second substrate, (S)-ASA. Yet, this finding is based on the assumption that (S)-lysine is a fully uncompetitive inhibitor. In light of crystallographic studies, which lead to the proposal that (S)-lysine affects the putative proton-relay of DHDPS, we re-evaluated the inhibition mechanism of DHDPS with respect to (S)-lysine by incorporating the observed hyperbolic inhibition. Our data showed that lysine is not an uncompetitive inhibitor, but a mixed inhibitor when pyruvate and (S)-lysine concentrations were varied. Thus, consistent with the crystallographic data, (S)-lysine must have an effect on the initial steps of the DHDPS reaction, including the binding of pyruvate and Schiff base formation.

Introduction

Dihydrodipicolinate synthase (DHDPS) mediates the key step in (S)-lysine biosynthesis. The reaction occurs at the branchpoint in the pathway and is feedback regulated by (S)-lysine. Since it was first characterised [1], DHDPS has attracted sustained interest in the literature. DHDPS is expressed in plants and micro-organisms, but not in animals, and thus attracts continued attention as a target for antibiotics and herbicides [2], [3], although no potent inhibitor has yet been found. As the purported rate-determining step in (S)-lysine biosynthesis, DHDPS also attracts the attention of biotechnologists aiming to engineer crops rich in (S)-lysine, often the limiting nutrient in staple crops [4]. That neither field of endeavour has yet yielded significant results may be due to our lack of fundamental knowledge of the enzyme mechanism.

DHDPS catalyses the condensation of (S)-aspartate-β-semialdehyde ((S)-ASA) and pyruvate to form an unstable heterocyclic product, currently thought to be (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTPA) (Scheme 1). This apparently simple condensation reaction is more complex than it first appears. In aqueous solution, (S)-ASA is known to exist predominantly in the hydrated form, rather than the aldehyde [5], [6], while the biologically relevant form of the substrate remains to be determined. There is also no direct evidence for the structure of the product of the enzyme, since no independent synthesis for this molecule has been reported. These chemical ambiguities have rendered a mechanistic analysis of DHDPS as very challenging.

The currently accepted mechanism of DHDPS [7] is outlined in Scheme 2, in which the structure of (S)-ASA is presumed to be the hydrate. In the first step of the mechanism, the active site lysine (Lys161 in Escherichia coli DHDPS) forms a Schiff base with pyruvate, as has been unequivocally demonstrated in several studies [8], [9], [10]. Subsequent binding of the second substrate—(S)-ASA—is followed by dehydration and cyclisation to form the product. Based on the X-ray crystal structure of the E. coli enzyme [7], [11], sequence homologies with DHDPS from other sources [12], and site-directed mutagenesis studies [13], it is proposed that a catalytic triad of three residues, tyrosine 133, threonine 44, and tyrosine 107 (E. coli numbering), act as a proton-relay to transfer protons to (and from) the active site via a water filled channel leading to the (S)-lysine binding site and bulk solvent.

DHDPS has been rigorously characterised in terms of enzyme kinetics by Karsten and others [1], [2], [14], who demonstrated that DHDPS was inhibited by (S)-lysine; the kinetic mechanism was found to be uncompetitive with respect to pyruvate and non-competitive with respect to (S)-ASA. However, these workers used pure inhibition models in order to simplify the analysis, in light of the observation that DHDPS exhibits partial inhibition, whereby residual activity is observed even at saturating inhibitor concentrations; this is also known as hyperbolic inhibition [15], [16]. If (S)-lysine asserts its influence by attenuating the function of the proton-relay, as proposed by Blickling et al. [7] and us [13], then given the likely role of this motif in pyruvate binding and Schiff base formation (Scheme 2), it seems unlikely that, as proposed by Karsten [14], (S)-lysine would affect the second half reaction only. To reconcile these seemingly conflicting results, we repeated the kinetic analysis of DHDPS with respect to (S)-lysine inhibition using the coupled assay and re-evaluated the data in terms of partial inhibition.

Section snippets

Chemicals

Unless otherwise stated, all chemicals were obtained from Sigma Chemical Company or Pharmacia. Protein concentration was measured by the method of Bradford [17]. Enzymes were manipulated at 4 °C, or on ice, unless otherwise stated, and were stored in Tris–HCl buffer (20 mM) at –20 °C. (S)-ASA was synthesised using the methods of Roberts et al. [18] and was of the highest quality (>95%), as judged by 1H NMR and the coupled assay. This synthesis was chosen to avoid aberrant complications of

Over-expression and purification of DHDPS

Wild-type DHDPS was over expressed in, and purified from E. coli XL-1 Blue cells according to the method of Mirwaldt et al. [9], [11] with modifications. For purification purposes, the o-aminobenzaldehyde assay was employed [1]. The protein was homogeneous as judged by SDS-PAGE [19] with Coomassie brilliant blue staining. Table 1 shows that the wild-type enzyme was purified nearly sixfold to a specific activity of 1.8 units mg–1. For kinetic characterisation we employed a coupled assay

Discussion

We have assessed the steady-state properties of DHDPS using the coupled assay. The kinetic mechanism was confirmed to be of the ping-pong type, while the kinetic parameters were consistent with those published elsewhere.

From X-ray crystallographic studies, the lysine binding site of DHDPS has been shown to be distal from the active site [7]. However, the mechanism by which (S)-lysine attenuates DHDPS function is not well understood. A thorough kinetic study by others [14] concluded that (S

Acknowledgements

This work was funded, in part, by Crop and Food Research Ltd. as part of contract C02X0001 for the New Economy Research Fund, Foundation for Research Science and Technology, New Zealand, and, in part, by the Royal Society of New Zealand Marsden Fund (Contract UOC303). The authors acknowledge Jennifer Turner and Craig Hutton (University of Sydney, Australia), Antonia Miller, Laurence Antonio, and Jane Allison (University of Canterbury, New Zealand) for useful discussions, materials, and

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