Substrate-mediated Stabilization of a Tetrameric Drug Target Reveals Achilles Heel in Anthrax

Jarrod E Voss,Stephen W Scally,Nicole L Taylor,Sarah C Atkinson,Michael D.W Griffin,Craig A Hutton,Michael W Parker,Malcolm R Alderton,Juliet A Gerrard,Renwick C.J Dobson,Con Dogovski,Matthew A Perugini

doi:10.1074/jbc.m109.038166

Abstract

Bacillus anthracis is a gram-positive spore-forming bacterium that causes anthrax. With the increased threat of anthrax in biowarfare, there is an urgent need to characterize new antimicrobial targets from B. anthracis. One such target is dihydrodipicolinate synthase (DHDPS), which catalyzes the committed step in the pathway yielding meso-diaminopimelate and lysine. In this study, we employed CD spectroscopy to demonstrate that the thermostability of DHDPS from B. anthracis (Ba-DHDPS) is significantly enhanced in the presence of the substrate, pyruvate. Analytical ultracentrifugation studies show that the tetramer-dimer dissociation constant of the enzyme is 3-fold tighter in the presence of pyruvate compared with the apo form. To examine the significance of this substrate-mediated stabilization phenomenon, a dimeric mutant of Ba-DHDPS (L170E/G191E) was generated and shown to have markedly reduced activity compared with the wild-type tetramer. This demonstrates that the substrate, pyruvate, stabilizes the active form of the enzyme. We next determined the high resolution (2.15 A) crystal structure of Ba-DHDPS in complex with pyruvate (3HIJ) and compared this to the apo structure (1XL9). Structural analyses show that there is a significant (91 A(2)) increase in buried surface area at the tetramerization interface of the pyruvate-bound structure. This study describes a new mechanism for stabilization of the active oligomeric form of an antibiotic target from B. anthracis and reveals an "Achilles heel" that can be exploited in structure-based drug design.

Highlights

That Bacillus anthracis is the causative agent of the disease
Effect of Pyruvate on the Secondary Structure Stability of BaDHDPS—We recently reported that the raw enzyme activity and yield of recombinant Ba-dihydrodipicolinate synthase (DHDPS) are significantly increased when the enzyme using a 4-hole An-60 Ti or an 8-hole An-50 Ti rotor
To sector quartz cells were loaded with 380 ␮l of sample and 400 ␮l examine the effect of pyruvate on the stability of Ba-DHDPS in of reference (20 mM Tris, 150 mM NaCl, pH 8.0) for sedimenta- aqueous solution, thermal denaturation experiments monition velocity, or a 100-␮l sample and a 120-␮l reference for tored by CD spectroscopy were conducted in the presence and sedimentation equilibrium

Summary

Introduction

That Bacillus anthracis is the causative agent of the disease. Given the tolerance of its spores to extreme physical conditions and their ease of dissemination, the use of B. anthracis in bioterrorism and biological warfare is a threat to developed countries worldwide. One potential therapeutic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the first committed step in the lysine biosynthetic pathway (Fig. 1A). Targeting the DAP pathway through disrupting the first committed step, DHDPS, will yield a novel class of therapeutic agents to treat anthrax. This notion is supported by recent work [7] that identified dapA, the gene encoding DHDPS from B. subtilis, as one of only 271 genes essential for cell viability from a total of Ͼ4,100 genes encoded by its genome. Mammals do not synthesize lysine and do not possess the DHDPS enzyme This suggests that specific inhibitors of DHDPS would have selective antibacterial activity with low toxicity in a mammalian host. We report solution and structural studies that unravel the mechanism for substrate-mediated stabilization of the active quaternary structure of Ba-DHDPS

Objectives

Results

Conclusion