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We report the atomic-resolution (1.3 Å) X-ray crystal structure of an open conformation of the dapE -encoded N- succinyl-l,l-diaminopimelic acid desuccinylase (DapE, EC 3.5.1.18) from Neisseria meningitidis . This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound sulfate ions in the active site that mimic the binding of the terminal carboxylates of the N- succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated inhibition of DapE by sulfate (IC 50 = 13.8 ± 2.8 mM). Comparison with other DapE structures in the PDB demonstrates the flexibility of the interdomain connections of this protein. This high-resolution structure was then utilized as the starting point for targeted molecular dynamics experiments revealing the conformational change from the open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated closure from the open to the closed conformation, the change in RMS throughout the closure, and the independence in the movement of the two DapE subunits. This conformational change occurred in two phases with the catalytic domains moving toward the dimerization domains first, followed by a rotation of catalytic domains relative to the dimerization domains. Although there were no targeting forces, the substrate moved closer to the active site and bound more tightly during the closure event.

Inhibitors of the bacterial enzyme dapE-encoded N -succinyl-L,L-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG)., Competing Interests: The authors declare no conflict of interest.

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.