L-Captopril and its derivatives as potential inhibitors of microbial enzyme DapE: A combined approach of drug repurposing and similarity screening.

The perils of antimicrobial drug resistance can be overcome by finding novel antibiotic targets and corresponding small molecule inhibitors. Microbial enzyme DapE is a promising antibiotic target due to its importance to the bacterial survival. The potency of L-Captopril, a well known angiotensin-converting enzyme inhibitor, as an inhibitor of DapE enzyme has been evaluated by analyzing its binding modes and binding affinity towards DapE enzyme. L-Captopril is found to bind the metal centers of DapE enzyme either via its thiolate group or through its carboxylate group. While the latter binding mode is found to be thermodynamically favorable, the former binding mode, also seen in the crystal structure, is kinetically favored. To optimize the binding affinity of the inhibitor towards DapE enzyme, a series of L-Captopril-based inhibitors have been modelled by changing the side groups of L-Captopril. The introduction of a bipolar functional group at the C4 position of the pyrrolidine ring of L-Captopril and the substitution of the thiol group with a carboxylate group, have been shown to provide excellent enzyme affinity that supersedes the binding affinity of DapE enzyme towards its natural substrate, thus making this molecule a potential inhibitor with great promise.

Active Site Dynamics in Substrate Hydrolysis Catalyzed by DapE Enzyme and Its Mutants from Hybrid QM/MM-Molecular Dynamics Simulation.

The mechanism of the catalytic hydrolysis of N-succinyl diaminopimelic acid (SDAP) by the microbial enzyme DapE in its wild-type (wt) form as well as three of its mutants (E134D, H67A, and H349A) is investigated employing a hybrid quantum mechanics/molecular mechanics (QM/MM) method coupled with molecular dynamics (MD) simulations, wherein the time evolution of the atoms of the QM and MM regions are obtained from the forces acting on the individual atoms. The free-energy profiles along the reaction coordinates of this multistep hydrolysis reaction process are explored using a combination of equilibrium and nonequilibrium (umbrella sampling) QM/MM-MD simulation techniques. In the enzyme-substrate complexes of wt-DapE and the E134D mutant, nucleophilic attack is found to be the rate-determining step involving a barrier of 15.3 and 21.5 kcal/mol, respectively, which satisfactorily explains the free energy of activation obtained from kinetic experiments in wt-DapE-SDAP (15.2 kcal/mol) and the 3 orders of magnitude decrease in the catalytic activity due to E134D mutation. The catalysis is found to be quenched in the H67A and H349A mutants of DapE due to conformational rearrangement in the active site induced by the absence of the active site His residues that prohibits activation of the catalytic water molecule.

Loss of Catalytic Activity in the E134D, H67A, and H349A Mutants of DapE: Mechanistic Analysis with QM/MM Investigation.

In the fight against bacterial infections and antibiotic resistance, the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a potentially safe target enzyme. The role of the Glu134, His67, and His349 residues in the binding and hydrolysis of N-succinyl-l,l-diaminopimelic acid (SDAP) is investigated by employing molecular dynamics simulation and hybrid quantum mechanical-molecular mechanical (MM) calculations of the E134D, H67A, and H349A mutants of DapE. The free energy of substrate binding obtained from the MM-Poisson-Boltzmann surface area approach correctly reproduced the experimentally observed ordering of substrate affinity, that is, E134D > wt > H67A > H349A. The mechanism of catalytic action by the E134D mutant is elucidated by structurally and energetically characterizing the intermediates and the transition states along the reaction pathway. The rate-determining step in the general acid-base hydrolysis reaction by the E134D mutant is found to be the nucleophilic attack step, which involves an activation energy barrier 10 kcal/mol greater than that in the wild-type (wt)-DapE. This explains the 3 orders of magnitude decrease in the experimentally determined kcat value for the E134D mutant compared to that of wt-DapE. In the H67A and H349A mutants, the Glu134 residue undergoes a conformational change and exhibits a strong coordination with the metal centers. This not only results in a weaker substrate binding in the two histidine mutants but also hinders the activation of the catalytic water molecule, which constitutes the first step of the substrate hydrolysis by DapE. This leads to an effective quenching of the catalytic activity in the H67A and H349A mutants.

Structural and mechanistic insight into substrate binding from the conformational dynamics in apo and substrate-bound DapE enzyme.

Conformational dynamics in large biomolecular systems is often associated with their physiological roles. The dynamics of a dimeric microbial enzyme, DapE, with great potential as an antibiotic target, has been studied employing long molecular dynamics simulations of the enzyme in apo form and in substrate bound complex form. The essential dynamics of the apo enzyme and the enzyme-substrate complex are extracted from the principal component analysis of the simulations of these two systems where the first two principal components are analyzed in detail. The essential motion of the enzyme in the substrate bound form exhibits a folding motion of its two catalytic domains over the two dimerization domains, which results in repulsion of water molecules away from the active site of the enzyme-substrate complex. This folding motion also leads to a stabilizing binding free energy of the substrate arising from the favorable interaction of the substrate and side chains of the enzyme. The dynamics in the enzyme-substrate complex results in stronger interaction between the catalytic and dimerization domains reflected by an increased number of inter-domain hydrogen bonds. The substrate, located in the catalytic domain of DapE, establishes contacts with the side chains of the dimerization domain of DapE by extended chains of hydrogen bonds, which emphasizes the role of the dimerization domain in substrate binding.

The structural and energetic aspects of substrate binding and the mechanism of action of the DapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) investigated using a hybrid QM/MM method.

With increasing cases of fatal bacterial infections and growing antibiotic resistance, unrelenting efforts are necessary for identification of novel antibiotic targets and new drug molecules. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a di-nuclear Zn containing enzyme in the lysine biosynthetic pathway which is indispensable for bacterial survival and absent in the human host, thus a potential antibiotic target. The DapE enzyme catalyzes the hydrolysis of N-succinyl-L,L-diaminopimelic acid (SDAP) to give rise to succinic acid and L,L-diaminopimelic acid. The mechanism of action of the DapE catalyzed SDAP hydrolysis is investigated employing a hybrid QM/MM computational method. The DapE side chains, such as, Arg178, Thr325, Asn345, are found to play a role in substrate identification and stabilization of the enzyme active site. Furthermore, a glycine rich loop (Gly322-Ser326) is found to facilitate tight binding of the substrate in the enzyme active site. The catalytic reaction progresses via a general acid-base hydrolysis mechanism where Glu134 first acts as a Lewis base by activating the catalytic water molecule in the active site, followed by guiding the resulting hydroxyl ion for a nucleophilic attack on the substrate, and finally acts as a Lewis acid by donating a proton to the substrate. The intermediates and transition states along the reaction pathway have been structurally and energetically characterized. A conformational change in the side chain of Asp100, which bridges the two Zn centers of the enzyme, is observed which facilitates the enzymatic action by lowering the activation energy and leads to the formation of a new intermediate during the catalytic reaction. The nucleophilic attack is found to be the rate determining step.

Quantum-mechanical DFT calculation supported Raman spectroscopic study of some amino acids in bovine insulin.

In this article Quantum mechanical (QM) calculations by Density Functional Theory (DFT) have been performed of all amino acids present in bovine insulin. Simulated Raman spectra of those amino acids are compared with their experimental spectra and the major bands are assigned. The results are in good agreement with experiment. We have also verified the DFT results with Quantum mechanical molecular mechanics (QM/MM) results for some amino acids. QM/MM results are very similar with the DFT results. Although the theoretical calculation of individual amino acids are feasible, but the calculated Raman spectrum of whole protein molecule is difficult or even quite impossible task, since it relies on lengthy and costly quantum-chemical computation. However, we have tried to simulate the Raman spectrum of whole protein by adding the proportionate contribution of the Raman spectra of each amino acid present in this protein. In DFT calculations, only the contributions of disulphide bonds between cysteines are included but the contribution of the peptide and hydrogen bonds have not been considered. We have recorded the Raman spectra of bovine insulin using micro-Raman set up. The experimental spectrum is found to be very similar with the resultant simulated Raman spectrum with some exceptions.

Structural diversity of copper(I) complexes formed by pyrrole- and dipyrrolylmethane-based diphosphine ligands with cu-X···HN hydrogen bonds.

Metal complexes containing hydrogen bond donor/acceptor groups are interesting because of their applications in several areas. In the course of our investigation on the synthesis of metal complexes using newly developed pyrrole-based diphosphine ligands, a few structurally interesting copper(I) complexes containing the pyrrolic NH hydrogen bond donors were synthesized. The reaction of 2,5-bis(diphenylphosphinomethyl)pyrrole (PNP) with an equimolar quantity of CuX (X = Cl, Br, and I) afforded the binuclear copper(I) complexes [Cu(µ-X)(µ-PNP-P,P)]2 (1-3) in very good yields (87-90%). Conversely, the analogous reaction between 1,9-bis(diphenylphosphinomethyl)diphenyldipyrrolylmethane (PNNP) and CuX (X = Cl, Br, and I) yielded the mononuclear Cu(I) complexes [CuX(PNNP-P,P)] (4-6) in very good yields (∼88%), in which the diphosphine ligand is chelated to the copper metal atom. Interestingly, when this reaction was carried out with a 1:2 mol ratio of ligand/metal, the cubane-like tetranuclear Cu(I) complex, [Cu4I4{µ-Ph2C(C4H3N)2-1,9-(CH2PPh2)2-P,P}2] 7, was isolated in 68% yield. In addition, the reaction between the dipyrrolyldiphosphine ligand (PNNP) and CuCl in the presence of 1 equiv of 1,10-phenanthroline monohydrate and NaBF4 afforded a novel ionic binuclear mixed-ligand Cu(I) complex, [Cu2(µ-X)(µ-PNNP-P,P)(NN)2]BF4 8, where NN = 1,10-phenanthroline in 57% yield. The structures of all these complexes were confirmed by the single-crystal X-ray diffraction method and are supported by spectroscopic data. In contrast to the PNP pincer ligand, the dipyrrolyl-diphosphine ligand (PNNP) adopts chelation as well as bridging coordination modes with Cu(I) atoms, indicating its flexibility of bonding. In all the structures, the Cu-X···HN type of hydrogen bonds involving the metal halide ion as acceptor and the pyrrolic NH as donor are present with the Cu-X···H angles, which deviate from the favored 90°, as observed in their solid state structures. Further, the presence of this type of hydrogen bond was confirmed by NBO, AIM, and Hirshfeld analyses.