The Dimerization Domain in DapE Enzymes Is Required for Catalysis

The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,Ldiaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate

The Role of the Dimerization Domain in the Stabilization of Loop V in WT-<i>Hi</i>DapE.

<p>A) Superimposition of the WT-<i>Hi</i>DapE (gray) and <i>Vc</i>DapE^T (cyan) structures is shown. Loop V of WT-<i>Hi</i>DapE and <i>Vc</i>DapE^T is labeled as <i>Hi</i>LV and <i>Vc</i>LV, respectively. WT-<i>Hi</i>DapE residues interacting with the sulfate ion (stick model) are shown as gray lines. Corresponding residues in <i>Vc</i>DapE^T (except for R258 that is absent in the deletion mutant) are shown as yellow (R179 and R180) and orange (G214) lines. B) Specific orientation of the active site loop V in <i>Vc</i>DapE^T and the corresponding loop in AAP. Overlay of the <i>Vc</i>DapE^T (cyan) and AAP (purple) structures is shown. The AAP loop and <i>Vc</i>DapE^T loop V are labeled as <i>Ap</i>LV and <i>Vc</i>LV, respectively. Stabilization of loop V in AAP by a disulfide bridge is indicated where Cys223 and Cys227 of AAP and the residues involved in zinc-binding in <i>Vc</i>DapE^T are shown as sticks. Zinc ions of <i>Vc</i>DapE^T are shown as black spheres. Zinc-bound ethylene glycol was omitted for clarity.</p

Diagrams Showing Regions of Flexibility in Truncated DapE Proteins.

<p>A) MOLMOL diagram of [ZnZn(<i>Vc</i>DapE^T)] molecular dynamics. B) MOLMOL diagram of [ZnZn(<i>Hi</i>DapE^T)] molecular dynamics (the thickness of the line is proportional to the variation of the protein structure during the simulation). The crystallographic temperature factors indicating that the most dynamic (in red) and the most rigid (in blue) parts of the protein: C) [ZnZn(<i>Vc</i>DapE^T)]. D) [ZnZn(<i>Hi</i>DapE^T)].</p

The Active Site of WT-<i>Hi</i>DapE Showing Loop V.

<p>T325 resides on loop V directly over the dinuclear active site.</p

Dynamic light scattering data.

<p><i>R</i>_H – hydrodynamic radius; MW – molecular weight; Pd – the polydispersity, or width of the distribution, in nm, determined using a cumulants analysis (where the data are fit to an assumed distribution of particle sizes and the average radius and spread of radii is reported); %Pd – defined as Pd/<i>R</i>_H, the polydispersity divided by the estimated hydrodynamic radius from the cumulants fit of the autocorrelation function multiplied by 100; Baseline – The measured value of the normalized intensity autocorrelation curve. Values of 1.000 indicate that the measured correlation curve has returned to the baseline within the defined time. Deviations from the theoretical value of 1.000 typically indicate a noisy baseline; SOS error- the sum of squares difference between the measured correlation curve and the best fit curve calculated using the cumulants method of analysis, where a dust and noise free monomodal (single distribution) low polydispersity (narrow distribution) sample is assumed.</p

Molecular Dynamic Simulation Showing Regions of Flexibility in Catalytic Domain.

<p>A) [ZnZn(<i>Vc</i>DapE)]. B) [ZnZn(<i>Hi</i>DapE)]. C) AAP. The thickness of the line is proportional to the variation of the protein structure during the simulation. AS indicates the active site area, LVeq. equivalent of the LV loop in <i>Hi</i>DapE).</p

Data and Refinement Statistics.

a<p><i>R</i>_merge = Σ<i>_hkl</i>Σ<i>_i</i>|<i>Ii</i>₍<i>hkl</i>₎−〈<i>I_hkl</i>〉|/Σ<i>_hkl</i>Σ<i>_iI_i</i>_(<i>hkl</i>), where I<i>i</i>₍<i>hkl</i>₎ is the <i>i</i>th observation of reflection <i>hkl</i>, and 〈<i>I_hkl</i>〉 is the weighted average intensity for all observations <i>i</i> of reflection <i>hkl</i>.</p><p>, and .</p>b<p>Numbers in parentheses are values for the highest-resolution bin.</p>c<p>As defined by MOLPROBITY (M.F. –the most favored/A.A additionally allowed).</p

<i>Bacillus anthracis</i> Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion‑, Substrate‑, and Product-Bound Complexes

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of <i>Bacillus anthracis</i> IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known <i>Cryptosporidium parvum</i> IMPDH inhibitors was examined for the <i>B. anthracis</i> enzyme and compared with those of three bacterial IMPDHs from <i>Campylobacter jejuni</i>, <i>Clostridium perfringens</i>, and <i>Vibrio cholerae</i>. The structures contribute to the characterization of the active site and design of inhibitors that specifically target <i>B. anthracis</i> and other microbial IMPDH enzymes