Biomimetic Design Results in a Potent Allosteric Inhibitor of Dihydrodipicolinate Synthase from Campylobacter jejuni Yulia V. Skovpen, Cuylar J. T. Conly, David A. R. Sanders,* and David R. J. Palmer* Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada S7N 5C9 * S Supporting Information ABSTRACT: Dihydrodipicolinate synthase (DHDPS), an en- zyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipi- colinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein- inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named “bislysine”, resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a K i near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development. ■ INTRODUCTION The need for novel antibiotics is well-documented; in particular, there has been a striking lack of new drugs for Gram-negative bacteria in the last three decades. 1 Dihydrodi- picolinate synthase (DHDPS) is considered a target for new antibiotics due to its essential role in the diaminopimelate (dap) pathway responsible for biosynthesis of meso-diaminopi- melate and L-lysine, required components of the cell wall peptidoglycan. 2,3 The chemical and kinetic mechanisms of DHDPS are well-described: DHDPS is a Schiff-base-dependent aldolase catalyzing the conversion of pyruvate and (S)- aspartate-β-semialdehyde (ASA) to (4S)-hydroxy-2,3,4,5-tetra- hydro-(2S)-dipicolinate, which spontaneously dehydrates to dihydrodipicolinate (Figure 1). 4 The reaction follows classical ping-pong kinetics, with pyruvate required to bind and form a Schiff base with an active-site lysine before ASA binds and reacts. 4,5 Despite this knowledge, inhibitors designed to target the active site of DHDPS have not yielded promising results, 6-9 and it has not been demonstrated that this enzyme can be inhibited effectively. Here we report that potent inhibition is possible, by mimicking the natural regulation mechanism. DHDPSs from Gram-negative bacteria are regulated by the end-product of the dap pathway, L-lysine, by allosteric binding at low concentrations of inhibitor. The DHDPS from Campylobacter jejuni (CjDHDPS), for example, is inhibited with an apparent IC 50 of 65 μM. 5 Complete analysis shows the inhibition behavior to be complex: lysine is an uncompetitive partial inhibitor with respect to the first substrate, pyruvate, and a mixed partial inhibitor with respect to ASA. The partial inhibition means that ∼10% of the activity remains at saturating concentrations of lysine. The inhibition is also highly cooperative. Structural studies help explain the observed cooperativity. DHDPS from Gram-negative bacteria (and many Gram- positives) are homotetrameric, and can be described as a loose dimer of tight dimers. At the tight dimer interface, an allosteric cavity binds two lysine molecules with their α-carbon Received: December 4, 2015 Published: February 2, 2016 Figure 1. Reaction catalyzed by DHDPS. Pyruvate condenses with Lys166 prior to binding by ASA and subsequent aldol reaction. Article pubs.acs.org/JACS © 2016 American Chemical Society 2014 DOI: 10.1021/jacs.5b12695 J. Am. Chem. Soc. 2016, 138, 2014-2020