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.

The three-dimensional structure of N-succinyldiaminopimelate aminotransferase from Mycobacterium tuberculosis.

Inhibitors of the enzymes of the lysine biosynthetic pathway are considered promising lead compounds for the design of new antibacterial drugs, because the pathway appears to be indispensable for bacteria and because it is absent in humans. As part of our efforts to structurally characterize all enzymes of this pathway in Mycobacterium tuberculosis (Mtb), we have determined the three-dimensional structure of N-succinyldiaminopimelate aminotransferase (DapC, DAP-AT, Rv0858c) to a resolution of 2.0 A. This structure is the first DAP-AT structure reported to date. The orthorhombic crystals of Mtb-DAP-AT contain one functional dimer exhibiting C(2) symmetry in the asymmetric unit. The homodimer displays the typical S-shape of class I pyridoxal-5'-phosphate (PLP)-binding proteins. The two active sites of the dimer both feature an internal aldimine with the co-factor PLP covalently bound to the Lys232, although neither substrate nor co-factor had been added during protein production, purification and crystallization. Nine water molecules are conserved in the active site and form an intricate hydrogen-bonding network with the co-factor and the surrounding amino acid residues. Together with some residual difference electron density in the active site, this architecture permitted the building of external aldimine models of the enzyme with the substrates glutamate, the amine donor, and N-succinyl-2-amino-6-keto-pimelate, the amine acceptor. Based on these models, the amino acids relevant for substrate binding and specificity can be postulated. Furthermore, in the external aldimine model of N-succinyl-2-amino-6-keto-pimelate, the succinyl group overlaps with a glycerol binding site that has also been identified in both active sites of the Mtb-DAP-AT dimer. A comparison of the structure of Mtb-DAP-AT with other class I PLP-binding proteins, revealed that some inhibitors utilize the same binding site. Thus, the proposed models also provide an explanation for the mode of inhibition of Mtb-DAP-AT and they may be of help in the design of compounds, which are capable of inhibiting the enzyme. Last, but not least, a chloride binding helix exhibiting a peculiar amino acid sequence with a number of exposed hydrophobic side-chains was identified, which may be hypothesized as a putative docking site.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapC (Rv0858c) from Mycobacterium tuberculosis.

N-Succinyldiaminopimelate aminotransferase from Mycobacterium tuberculosis (DAP-AT; DapC; Rv0858c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in two related crystal forms. Preliminary diffraction data analysis suggests the presence of a monomer in the asymmetric unit of the tetragonal crystal form and a dimer in the asymmetric unit of the orthorhombic crystal form.