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

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 Å ) 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 Å 2 ) increase in buried surface area at the tetramerization interface of the pyruvatebound 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.

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

“…Interestingly, these interactions are also observed for several other enzymes, including dihydrodipicolinate synthase, which catalyzes the first committed and rate-limiting step in the DAP biosynthesis pathway (2,3). For dihydrodipicolinate synthase, dimerization has been shown to be important for completing the active site of each monomer through the interdigitation of a key catalytic triad residue (18,20,22,23,35,40). Similarly, recent studies demonstrate that dimerization is required for the catalytic activity of diaminopimelate epimerase, which also functions in the DAP pathway (16).…”

“…7C). A distinct upward curvature is observed between 5 and 500 nM, which is indicative of a self-associating enzyme where the lower order species is significantly less active than the higher order oligomer (16,20,23). By contrast, the wild-type enzyme displays a linear rate versus concentration relationship at concentrations (5-50 nM) well above the K D 231 (Fig.…”

Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogenLegionella pneumophila

“…The structure of DHDPS has been investigated in several bacteria including Agrobacterium tumefaciens , Bacillus anthracis (Blagova et al, 2006;Voss et al, 2009Voss et al, , 2010, Clostridium botulinum , Corynebacterium glutamicum (Rice et al, 2008), Escherichia coli (Mirwaldt et al, 1995;Dobson, Griffin et al, 2005), Hahella chejuensis (Kang et al, 2010), Methanococcus jannaschii (Padmanabhan et al, 2009), Mycobacterium tuberculosis (Kefala et al, 2008;Evans et al, 2011), Neisseria meningitidis (Devenish et al, 2009), Pseudomonas aeruginosa (Kaur et al, 2011), Shewenella benthica (Wubben et al, 2010), Staphylococcus aureus (Burgess et al, 2008;Girish et al, 2008), Streptococcus pneumoniae (Sibarani et al, 2010) and Thermotoga maritima (Pearce et al, 2006). The enzyme typically forms a homotetramer that is described as a dimer of dimers.…”

“…Reports of the pyruvatestabilization phenomenon (Blickling, Renner et al,1997;Burgess et al, 2008;Voss et al, 2010;Atkinson et al, 2009) prompted cocrystallization of Lp-DHDPS with pyruvate. Diffraction was found to significantly improve with the substrate-bound form of the enzyme.…”

Enzymology of Bacterial Lysine Biosynthesis

“…1), yields the important metabolites meso-2,6-diaminopimelate (meso-DAP) and lysine. Lysine is utilised for protein synthesis in bacteria and forms part of the peptidoglycan cross-link structure in the cell wall of most Gram-positive species; whilst meso-DAP is the peptidoglycan cross-linking moiety in the cell wall of Gram-negative bacteria and also Gram-positive Bacillus species Mitsakos et al, 2008;Voss et al, 2010) (Fig. 1).…”