A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

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Abstract

Dihydrodipicolinate synthase (DHDPS) is a validated antibiotic target for which a new approach to inhibitor design has been proposed: disrupting native tetramer formation by targeting the dimer–dimer interface. In this study, rational design afforded a variant of Mycobacterium tuberculosis, Mtb-DHDPS-A204R, with disrupted quaternary structure. X-ray crystallography (at a resolution of 2.1 Å) revealed a dimeric protein with an identical fold and active-site structure to the tetrameric wild-type enzyme. Analytical ultracentrifugation confirmed the dimeric structure in solution, yet the dimeric mutant has similar activity to the wild-type enzyme. Although the affinity for both substrates was somewhat decreased, the high catalytic competency of the enzyme was surprising in the light of previous results showing that dimeric variants of the Escherichia coli and Bacillus anthracis DHDPS enzymes have dramatically reduced activity compared to their wild-type tetrameric counterparts. These results suggest that Mtb-DHDPS-A204R is similar to the natively dimeric enzyme from Staphylococcus aureus, and highlight our incomplete understanding of the role played by oligomerisation in relating protein structure and function.

Highlights

► A dimeric variant of DHDPS from Mycobacterium tuberculosis was engineered. ► The tertiary fold was preserved, but the quaternary structure was fully disrupted. ► Surprisingly, the activity was essentially conserved.

Introduction

Mycobacterium tuberculosis causes more death than any other bacterium [1], a problem that is increasing with the emergence of multi-drug resistant strains [2]. Accordingly, there is an urgent need to develop new anti-tuberculosis drugs and characterise novel drug targets. One such target is dihydrodipicolinate synthase (DHDPS)1 [3], an enzyme necessary for bacterial growth, which catalyses a key step in the metabolic pathway yielding the essential amino acid (S)-lysine and meso-diaminopimelic acid (DAP) [4], which is of particular importance in the mycobacterial cell wall [5]. DAP auxotrophs of Mycobacterium smegmatis have been observed to undergo cell death or show increased susceptibility to antibiotics in the absence of DAP [6], [7], [8]. In addition, M. tuberculosis has a demonstrated inability to utilise environmental (S)-lysine [9], suggesting the bacteria would be particularly vulnerable to inhibitors of DHDPS.

DHDPS catalyses the condensation of pyruvate and (S)-aspartate semi-aldehyde [(S)-ASA] to form an unstable heterocyclic product, 4-hydroxytetrahydrodipicolinate (HTPA) [10], [11] (Fig. 1, [12]). Of the various DHDPS enzymes characterised to date, all but one is tetrameric, with a dimer of dimers arrangement [12], [13], [14]. The only native DHDPS found to have a non-tetrameric quaternary structure is the dimeric Staphylococcus aureus DHDPS (Sa-DHDPS) [15], [16], indicating that the homotetrameric quaternary structure is not essential for full activity in all orthologues [15], [16]. All wild-type DHDPS feature a dimerisation interface formed by face-to-face contact of a pair of (βα)8 TIM barrel subunits. The second tetramerisation interface is formed by side-by-side contact of pairs of barrels, leading to a distinctive doughnut-shaped tetramer, with the exception reported for the plant DHDPS from Nicotiana sylvestris. The importance of the tetrameric structure of Escherichia coli DHDPS (Eco-DHDPS) has previously been demonstrated: dimeric variants of Eco-DHDPS, created by mutating one of the residues involved in inter-subunit interactions, showed significantly less activity than the tetrameric wild-type enzyme [17], [18] and quaternary structure has more recently been demonstrated to be important in Bacillus anthracis DHDPS (Ba-DHDPS) [19]. Thus, we predicted that the tetrameric structure would also be important for the activity of Mtb-DHDPS and sought to verify the dimer–dimer interface as a new potential target for antibiotic design [20].

In a previous study, we reported the X-ray crystal structure and biochemical and biophysical characterisation of the wild-type Mtb-DHDPS. In particular, we established using analytical ultracentrifugation (AUC) that it was a stable tetramer, even at dilute concentrations [21]. The focus of this study was the disruption of the dimer-dimer interface to produce discrete dimeric subunits, analogous to the wild-type Sa-DHDPS dimer, to test the importance of quaternary structure in Mtb-DHDPS [14]. We describe herein the production and characterisation of a dimeric variant and a comparison of its biophysical and biochemical characteristics with the wild-type enzyme.

Section snippets

Site-directed mutagenesis

Mutations were introduced into the pETM11 plasmid containing the M. tuberculosis dapA gene (Rv2753c) [22] using the QuikChange site-directed mutagenesis kit (Stratagene) and the mutagenic oligonucleotide primers (Invitrogen) listed in Table S1. DNA sequencing confirmed the presence of mutation and the integrity of the full length dapA insert.

Protein expression and purification

The mutated plasmid was used to transform E. coli BL21(DE3) cells containing the pGroESL plasmid [23]. Cells were grown for 4 h at 37 °C and 12 h at room

Strategy for engineering dimeric Mtb-DHDPS variants

In designing mutations to disrupt the dimer-dimer interface of the homotetrameric Mtb-DHDPS, we focused on introducing repulsive forces at the interface, an approach that was successfully applied to Eco-DHDPS [17], [18] and Ba-DHDPS [19]. The number of hydrophobic residues, as identified using JavaProtein Dossier (JPD), was greater in Mtb-DHDPS than Eco-DHDPS (11 vs. 3). JPD [36] and EBI PISA [37], in combination with visual inspection of the crystal structure of wild-type Mtb-DHDPS (PDB entry:

Discussion

Given that previous investigations with dimeric mutants of Eco-DHDPS found a dramatic decrease in catalytic activity compared to the wild-type enzyme [17], [18], we predicted that a similar result would be obtained for Mtb-DHDPS-A204R, validating the dimer-dimer interface as a target for antibiotic action. The mutation of alanine to arginine at position 204 was sufficient to disrupt the dimer-dimer interface, and a dimeric quaternary structure was observed both in the crystal structure of Mtb

Acknowledgments

We thank Dr. Marie Squire for assistance with mass spectrometry; Assoc. Prof. Emily Parker for useful discussions; Jackie Healy for exquisite technical assistance; and Dr. Andrew Muscroft-Taylor for synthesis of the aspartate semi-aldehyde. SRAD is grateful to the Foundation for Research Science and Technology for postdoctoral funding (Contract UOCX0603). R.C.J.D. acknowledges the C.R. Roper and The Royal Society of New Zealand Marsden Fund (Contract UOC1013) for support and M.A.P. for Future

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    Coordinate and structure factor files for Mtb-DHDPS-A204R have been deposited in the RCSB Protein Data Bank (PDB ID: 3L21).

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