Novel inhibitors of dihydrodipicolinate synthase

Abstract

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of L-lysine and meso-diaminopimelate biosynthesis, which is the condensation of (S)-aspartate-β-semialdehyde (ASA) and pyruvate into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS has been an attractive antibiotic target because L-lysine and meso-diaminopimelate are cross-linking components between peptidoglycan heteropolysaccharide chains in bacterial cell walls. Studies revealed that mutant auxotrophs for diaminopimelate undergo lysis in the absence of diaminopimelate in the medium; therefore the assumption is that strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and L-lysine biosynthesis in bacteria and would stop or decrease bacterial growth (eventually leading to bacterial death). In this work, the DHDPS inhibitor design is focused on the allosteric site of the enzyme. It was proposed that a compound mimicking binding of two L-lysine molecules at the allosteric site at the enzyme’s dimer-dimer interface would be a more potent inhibitor than the natural allosteric inhibitor of this enzyme, L-lysine. This inhibitor (R,R-bislysine) was synthesized as a racemic mixture, which was then separated with the aid of chiral HPLC. The mechanism of feedback inhibition of DHDPS from Campylobacter jejuni with its natural allosteric modulator, L-lysine, and its synthetic mimic, R,R-bislysine, is studied in detail. It is found that L-lysine is a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. R,R-bislysine is a mixed partial inhibitor with respect to pyruvate and a noncompetitive partial inhibitor with respect to ASA, with an inhibition constant of 200 nM. Kinetic evaluation of each DHDPS mutants (Y110F, H56A, H56N, H59A and H59N) has revealed amino acids responsible for the inhibitory effect of L-lysine, R,R-bislysine, and we have found that R,R-bislysine is a strong submicromolar inhibitor of Y110F, H56A, H56N and H59N.