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

Abstract

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.

On the Structure and Reaction Mechanism of Human Acireductone Dioxygenase

“…All residues with atoms within 15 from Fe were allowed to optimize their geometry,w hereas coordinates of the remaining part of the system were fixed (see Figure S1 in the Supporting Information). Previous works have shown that mechanical embedding with ac harge-update scheme converges very quickly [56] and yields reaction-energy profiles very similar to those obtained with electronic embedding. Twob asis sets were employed:t he lacvp basis (BS1) for most optimisations, frequency and ESP calculations and ab asis set that combined the lacv3p + basis set for the metal ion with 6-311G(d,p) basis set for all other atoms (BS2).…”

“…Finally, the geometry was reoptimised with the new set of QM charges. Previous works have shown that mechanical embedding with a charge‐update scheme converges very quickly and yields reaction‐energy profiles very similar to those obtained with electronic embedding . A D3(BJ) van der Waals correction was computed for the QM region with the DFTD3 program .…”

Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism

“…The amide carbonyl of l , l -SDAP is positioned near Zn2, while the amide N–H acts as a hydrogen bond donor to the backbone carbonyl of Thr325.A, a residue that was previously implicated as being catalytically important. 37 In addition, His194.B moves into the active site and forms an oxyanion hole along with Zn2, via a strong hydrogen bonding interaction with the amide carbonyl oxygen, O2. The proximal carboxylate of l , l -SDAP participates in bifurcated hydrogen bonds with side chains Arg258.A, Thr325.A, and Asn245.B (Figure 8B).…”

On the Difference Between Additive and Subtractive QM/MM Calculations

“…ELAC is implemented in the ONIOM software, but in practice it gives often severe convergence problems and is therefore seldom used. Instead, alternative approaches have been implemented, based on iterative calculations with mechanical embedding and updated charges (Kawatsu et al, 2011 ; Dutta and Mishra, 2014 ; Wójcik et al, 2014 ). It could therefore be recommended that ONIOM implements electrostatic embedding with only VLAC, which is a very stable approach in ComQum.…”