Exploring the dihydrodipicolinate synthase tetramer: How resilient is the dimer–dimer interface?

doi:10.1016/j.abb.2009.11.014

Archives of Biochemistry and Biophysics

Volume 494, Issue 1, 1 February 2010, Pages 58-63

https://doi.org/10.1016/j.abb.2009.11.014 Get rights and content

Abstract

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) is a tetrameric enzyme that catalyses the first committed step of the lysine biosynthetic pathway. Dimeric variants of DHDPS have impaired catalytic activity due to aberrant protein motions within the dimer unit. Thus, it is thought that the tetrameric structure functions to restrict these motions and optimise enzyme dynamics for catalysis. Despite the importance of dimer–dimer association, the interface between subunits of each dimer is small, accounting for only 4.3% of the total monomer surface area, and the structure of the interface is not conserved across species. We have probed the tolerance of dimer–dimer association to mutation by introducing amino acid substitutions within the interface. All point mutations resulted in destabilisation of the ‘dimer of dimers’ tetrameric structure. Both the position of the mutation in the interface and the physico-chemical nature of the substitution appeared to effect tetramerisation. Despite only weak destabilisation of the tetramer by some mutations, catalytic activity was reduced to ∼10–15% of the wild-type in all cases, suggesting that the dimer–dimer interface is finely tuned to optimise function.

Introduction

In the biosynthetic pathway leading to meso-diaminopimelate (DAP)¹ and (S)-lysine in plants and bacteria, dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) catalyses the branch point reaction: the condensation of (S)-aspartate-β-semialdehyde [(S)-ASA] and pyruvate to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. Since (S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design [1].

The crystal structures of a number of bacterial DHDPS enzymes [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13] and a plant enzyme [14] have been solved. With the exception of the recently reported dimeric Staphylococcus aureus enzyme [4], all functionally and structurally characterised DHDPS enzymes are homotetramers. The Escherichia coli DHDPS tetramer can be described as a dimer of tight-dimers (Fig. 1a), where there are many interactions between monomers A and B (or C and D), but relatively few between the two tight-dimers (A–B and C–D) [6]. Interestingly, the arrangement of the tight-dimers is different within the bacterial and plant tetramers, with dimer–dimer contacts formed on the opposite face of each subunit [3], [15].

Each subunit is composed of an (β/α)₈-barrel containing an active site, situated in the centre of the barrel, and each tight-dimer contains two complete active sites and an inhibitory-(S)-lysine binding site within the cleft between the subunits. A tyrosine residue from one subunit of the tight-dimer protrudes into the active site of the adjacent subunit and forms part of a catalytic triad that is essential for activity [16]. This suggests that the tight-dimer is the minimum unit necessary for DHDPS catalysis. However, mutation of a central residue in the dimer–dimer interface (L197) produced dimeric variants that displayed severely impaired catalytic function [15], due to aberrant dynamics occurring between the subunits of the dimer that were not present in the tetramer. These movements were proposed to be responsible for a reduction in substrate specificity that resulted in the covalent trapping of a substrate analogue, α-ketoglutarate, at the active site of the dimer. This suggests that the tetrameric structure of E. coli DHDPS evolved to optimise the dynamics within the tight-dimer unit.

In DHDPS from S. aureus, which occurs naturally as a dimer [4], the tight-dimer interface is significantly more extensive than in DHDPS from other species, in what is hypothesised to be an alternate evolutionary solution to optimising dynamics across this critical interface [4].

Strikingly, despite its role in enzyme function, the dimer–dimer interface of DHDPS is not conserved across species, suggesting that the key feature for function is the interface itself, rather than any specific amino acids. In this study, we probe the resilience of the dimer–dimer interface to mutation. We report that mutations at positions 193, 196, and 234, incorporating charge–charge repulsion (Q196D, Q234D), removal of hydrogen bonds (D193A, D193Y) or introducing steric bulk (D193Y), significantly attenuate the tetrameric quaternary structure and/or catalytic competency of E. coli DHDPS.

Section snippets

Materials and methods

All materials were obtained from Sigma–Aldrich. Enzymes were manipulated at 4 °C or on ice and were stored in 20 mM Tris–HCl, pH 8.0 at −20 °C.

The dimer–dimer interface of E. coli DHDPS is unusually small

The solvent inaccessible surface area at the dimer–dimer interface of E. coli DHDPS is 496 Å², which accounts for only 4.3% of the subunit surface area (11,551 Å²) (Fig. 1b). Furthermore, the total contact area between tight-dimers in the tetramer (992 Å²) is 4.8% of the total tight-dimer solvent accessible surface area (20,520 Å²). In a recent poll of 1494 interfaces within oligomeric proteins from the PDB [23], only 1.1% formed an interface of less than 5% of the total monomer surface area.

Discussion

Sedimentation studies clearly show that single point mutations introduced at the dimer–dimer interface, incorporating charge–charge repulsion, steric bulk or preventing hydrogen bonding at these positions, can significantly attenuate the ability of E. coli DHDPS to form the natural ‘dimer of dimers’ tetrameric structure in solution (Fig. 3 and Table 2). The mutant enzymes possessed significantly reduced catalytic activity (Table 3); however, these were completely folded (Fig. 2) and lysine

Acknowledgments

This work was funded by the Royal Society of New Zealand Marsden Fund (Contract UOC303); Crop & Food Research Ltd, Contract C02X0001 for the New Economy Research Fund, Foundation for Research Science and Technology, New Zealand; Defense Threat Reduction Agency, Project AB07CBT004, and the Australian Research Council, Project DP0770888 and Australian Postdoctoral Fellowship (M.A.P.). R.C.J.D. holds a C.R. Roper Fellowship. M.D.W.G. holds a Melbourne Early Career Researcher Grant. We thank Jackie

References (32)

B.R. Burgess et al.
J. Biol. Chem.
(2008)
S.R.A. Devenish et al.
Biochim. Biophys. Acta
(2009)
C. Mirwaldt et al.
J. Mol. Biol.
(1995)
E.A. Rice et al.
Arch. Biochem. Biophys.
(2008)
T.S. Girish et al.
FEBS Lett.
(2008)
M.D.W. Griffin et al.
J. Mol. Biol.
(2008)
R.C.J. Dobson et al.
J. Mol. Biol.
(2004)
P. Schuck et al.
Biophys. J.
(2002)
P.H. Brown et al.
Biophys. J.
(2006)
J. Vistica et al.
Anal. Biochem.
(2004)

M.J. Selwyn

Biochim. Biophys. Acta

(1965)

E. Krissinel et al.

J. Mol. Biol.

(2007)

J.G. Shedlarski et al.

J. Biol. Chem.

(1970)

R.C.J. Dobson et al.

Biochimie

(2004)

S.R.A. Devenish et al.

Biochem. Biophys. Res. Commun.

(2009)

C.A. Hutton et al.

Mol. Biosyst.

(2007)

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Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.
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Accordingly, DHDPS is considered a promising antibiotic target (Cox et al., 2000; Gerrard et al., 2007; Hutton et al., 2007). Previous studies suggest that the quaternary structure of DHDPS evolved to optimize protein dynamics for function (Burgess et al., 2008; Griffin et al., 2008, 2010; Muscroft-Taylor et al., 2010; Pearce et al., 2008, 2011; Reboul et al., 2012; Soares da Costa et al., 2015). Although dimeric forms of the enzyme have been described (Burgess et al., 2008; Girish et al., 2008; Kaur et al., 2011), structural studies demonstrate that DHDPS from plants (Atkinson et al., 2012, 2013; Blickling et al., 1997a; Griffin et al., 2012) and most bacteria (Atkinson et al., 2014; Blagova et al., 2006; Christensen et al., 2016; Devenish et al., 2009; Dobson et al., 2005b; Dogovski et al., 2009, 2012; 2013; Kang et al., 2010; Kefala et al., 2008; Mirwaldt et al., 1995; Padmanabhan et al., 2009; Pearce et al., 2006; Perugini et al., 2005; Phenix et al., 2008; Rice et al., 2008; Skovpen et al., 2016; Soares da Costa et al., 2015, 2016; Sowole et al., 2016; Sridharan et al., 2014; Voss et al., 2010; Wolterink-van Loo et al., 2008) assemble as a homotetramer (Figures 1B and 1C).
Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.
Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae
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Following confirmation that the recombinant proteins contain the correct primary structure, the secondary structures of untagged and His-tagged Vc-DHDPS were determined using CD spectroscopy. The CD spectra for both forms of Vc-DHDPS showed double minima spanning 208–225 nm (Fig. 3A), suggesting a mixed α/β fold as observed for other orthologs [5,7,17,26,27,45]. Indeed, nonlinear regression analyses confirm this assertion showing that both untagged and His-tagged Vc-DHDPS consist of 55% mixed α/β structure (Fig. 3A, solid lines).
Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS; EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, K_M (pyruvate) of 0.14 mM and 0.15 mM, K_M (L-aspartate-4-semialdehyde) of 0.08 mM and 0.09 mM, and k_cat of 34 and 46 s⁻¹, respectively. These results demonstrate there are no significant changes in the structure and function of Vc-DHDPS upon the addition of an N-terminal His tag and, hence, the tagged recombinant product is suitable for future studies, including screening for new inhibitors as potential anti-cholera agents. Additionally, a polyclonal antibody raised against untagged Vc-DHDPS is validated for specifically detecting recombinant and native forms of the enzyme.
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Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect. We subsequently employed our crystallographic data with bioinformatics, mutagenesis, enzyme kinetics, and microscale thermophoresis to reveal that lysine-mediated inhibition is not defined by Gram staining, but by the presence of a His or Glu at position 56 (Escherichia coli numbering). This study has unveiled the molecular determinants defining lysine-mediated allosteric inhibition of bacterial DHDPS.
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Here, we review recent studies aimed at defining the importance of quaternary structure to a model oligomeric enzyme, dihydrodipicolinate synthase. This will illustrate the complementary and synergistic outcomes of coupling the techniques of analytical ultracentrifugation with enzyme kinetics, in vitro mutagenesis, macromolecular crystallography, small angle X-ray scattering, and molecular dynamics simulations, to demonstrate the role of subunit self-association in facilitating protein dynamics and enzyme function. This multitechnique approach has yielded new insights into the molecular evolution of protein quaternary structure.
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As such, the enzyme is the product of an essential bacterial gene and is thus a valid target for novel drug discovery [4,7–11]. Consequently, there has been an increased interest in studying the enzymes of bacterial lysine biosynthesis, particularly in characterizing the kinetic properties, regulation, and three-dimensional structures of both wild-type and mutant enzymes [7–10,12–23]. In MRSA, DHDPR is the product of the dapB gene [7–11].
Given the rise of multi drug resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to discover new antimicrobial agents. A validated but as yet unexplored target for new antibiotics is dihydrodipicolinate reductase (DHDPR), an enzyme that catalyzes the second step of the lysine biosynthesis pathway in bacteria. We report here the cloning, expression and purification of N-terminally his-tagged recombinant DHDPR from MRSA (6H-MRSA–DHDPR) and compare its secondary and quaternary structure with the wild type (MRSA–DHDPR) enzyme. Comparative analyses demonstrate that recombinant 6H-MRSA–DHDPR is folded and adopts the native tetrameric quaternary structure in solution. Furthermore, kinetic studies show 6H-MRSA–DHDPR is functional, displaying parameters for $K_{m}^{NADH}$ of 6.0 μM, $K_{m}^{DHDP}$ of 22 μM, and k_cat of 21 s⁻¹, which are similar to those reported for the native enzyme. The solution properties and stability of the 6H-MRSA–DHDPR enzyme are also reported in varying physicochemical conditions.

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Original paperExploring the dihydrodipicolinate synthase tetramer: How resilient is the dimer–dimer interface?

Abstract

Introduction

Section snippets

Materials and methods

The dimer–dimer interface of E. coli DHDPS is unusually small

Discussion

Acknowledgments

J. Biol. Chem.

Biochim. Biophys. Acta

J. Mol. Biol.

Arch. Biochem. Biophys.

FEBS Lett.

J. Mol. Biol.

J. Mol. Biol.

Biophys. J.

Biophys. J.

Anal. Biochem.

Biochim. Biophys. Acta

J. Mol. Biol.

J. Biol. Chem.

Biochimie

Biochem. Biophys. Res. Commun.

Mol. Biosyst.

Original paper
Exploring the dihydrodipicolinate synthase tetramer: How resilient is the dimer–dimer interface?