UNDERSTANDING ALLOSTERIC INHIBITION MECHANISMS IN DIHYDRODIPICOLINATE SYNTHASE FROM CAMPYLOBACTER JEJUNI USING X-RAY CRYSTALLOGRAPHY AND B-FACTORS ANALYSIS

Abstract

Dihydrodipicolinate (DHDPS) synthase catalyzes the condensation of pyruvate and (S)-aspartate β-semialdehyde [(S)-ASA] to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTPA), which is decomposed into (S)-lysine and meso-diaminopimelate. Inhibition of DHDPS would lead to the disruption of meso-diaminopimelate resulting in no bacterial cell wall synthesis and eventually killing the bacteria. The absence of (S)-lysine biosynthesis in humans and the presence of meso-diaminopimelate and (S)-lysine in the bacterial cell wall makes DHDPS an attractive antibiotic target. Kinetic studies have shown that CjDHDPS is allosterically inhibited by L-lysine and R, R-bislysine. The crystal structures of wild-type Campylobacter jejuni (Cj) CjDHDPS have shown that lysine and bislysine bind at the tight-dimer interface and form hydrogen bonds with amino acid residues, without global conformational changes. Hydrogen/deuterium exchange (HDX)-mass spectrometry (HDX-MS) studies showed that lysine and bislysine increase the rigidity of the active site regions of the wild-type CjDHDPS, likely to prevent diffusion and correct binding of the substrates in the active site. Currently, the roles of tight dimer interface residues in the transmission of the allosteric inhibition signals in CjDHDPS are poorly understood. Based on this information, I hypothesize that lysine and bislysine allosterically inhibit CjDHDPS enzyme by altering enzymes flexibility critical for enzyme activity. The first research objective was to use B-factors from reported structures to correlate changes in B-factors with the HDX-MS studies to explain dynamic allosteric wild-type CjDHDPS mechanisms in terms of flexibility differences at an atomic level. The B-factor analysis showed rigidification of the active site regions upon lysine/bislysine binding, suggesting restriction of motions could prevent the diffusion/correct binding at the active site, consistent with our hypothesis and the published HDX-MS studies. The second research objective was to obtain high-resolution crystal structures of the CjDHDPS mutants (H56A, H56N, H56W, H59A, H59N, H59K, N84A, N84D, E88A, E88D, E88Q, and Y110F) with and without inhibitors and to use B-factors to identify structural features corresponding to the allosteric inhibition mechanisms upon inhibitor binding. The crystal structures showed that L-lysine and R, R-bislysine binds in the same configuration and participates in forming the same set of hydrogen bond interactions in all DHDPS mutants. There are no global and local conformational changes upon the inhibitors' binding except subtle differences in the hydrogen bond interaction pattern between the catalytic residues. The structural data suggested that the inhibitors may allosterically inhibit the enzyme by altering protein flexibility rather than conformational changes. B-factor analysis shows that bislysine allosterically increase the rigidity of the C-terminal active-site/weak dimer regions as compared to apo DHDPS and lysine bound DHDPS, to prevent the correct binding/diffusion of the substrate in the active site, suggesting a mechanism of allosteric inhibition across CjDHDPS mutants.