Biochemical and Structural Analysis of the Bacterial Enzyme Succinyl-Diaminopimelate Desuccinylase (DapE) from Acinetobacter baumannii

Abstract

There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae (HiDapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii (AbDapE) and studied inhibition by known inhibitors of HiDapE. AbDapE is inhibited by captopril and sulfate comparable to HiDapE, but AbDapE was not significantly inhibited by a known indoline sulfonamide HiDapE inhibitor. Captopril and sulfate both stabilize HiDapE by increasing the thermal melting temperature (T_m) in thermal shift assays. By contrast, sulfate decreases the stability of the AbDapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted AbDapE in the closed conformation, one with AbDapE in complex with succinate derived from enzymatic hydrolysis of N⁶-methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of AbDapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution).

Scheme 1. Hydrolysis of l,l-SDAP and a substrate analogue by DapE

l,l-SDAP endogenous substrate (1a) and assay substrate N⁶-methyl-l,l-SDAP7 (1b) with formation of hydrolysis products succinate (2) and l,l-diaminopimelic acid derivatives 3a and 3b, respectively.

Figure 1

The structure of A. baumannii DapE-SeMet. (A) Overall structure of the AbDapE-SeMet dimer from crystal 2 (PDB code 8F8O). Chain A is colored wheat, chain B yellow, and active site zinc ions are magenta. Atoms of the succinic acid (lime green carbons) and l-(S)-lactate (light gray carbons) are colored oxygen in red and nitrogen in blue. (B) Zoomed-in view of the zinc-binding site from chain A with atoms colored as in (A). Zinc-coordinating residues are shown as sticks. Atoms of succinic acid and l-(S)-lactate are also colored carbon in wheat, oxygen in red, and nitrogen in blue. (C) Superposition of the AbDapE-SeMet structure of crystal 2 (8F8O, chain A in wheat, chain B in yellow) and the structure of crystal 1 (7T1Q, chain A in teal, chain B in cyan) active sites. Succinic acid, l-(S)-lactate (8F8O, wheat carbons), and acetate (7T1Q, teal carbons) are shown, and zinc ions are shown as spheres in magenta (8F8O) and orange (7T1Q).

Scheme 2. Hypothesized X-ray Induced Loss of Acetate and CO₂ Forming Lactate in the Active Site

Figure 2

Close-up view of the active sites of superimposed structures of HiDapE (PDB 5VO3, chain A pale green, symmetry-related chain of the dimer in pale violet) and AbDapE (PDB 8F8O chain A wheat, chain B yellow). Residues His195 and Tyr198 of chain B from AbDapE and corresponding residues from HiDapE and products of the reactions are shown as sticks (carbons match the colors of the chains, oxygen in red, nitrogen in blue), and zinc shown as spheres (magenta for 8F8O and pink for 5VO3). Bonds between His195.B and Tyr198.B of AbDapE and oxygen atoms of the succinate are shown by dashed lines with distances provided in angstroms.

Figure 3

Closed and open states of DapE result from movement in the hinge region. Overlaps of (A) open NmDapE and HiDapE and (B) closed AbDapE structures. The distances between c-terminal residues of α-helix 3 are shown with dashed lines. (C) Superimposed catalytic and dimerization domains of AbDapE (7T1Q), HiDapE, and NmDapE. (D) Superimposed hinge regions of AbDapE (7T1Q), HiDapE, and NmDapE. The α-helix 7 of the regions was aligned to emphasize movement of hinge residues. Coloring scheme is the same as in panels A and B.