How the Substrate d-Glutamate Drives the Catalytic Action of Bacillus subtilis Glutamate Racemase

Abstract

Molecular Dynamics simulations with a Molecular Mechanics force field and a quite complete exploration of the QM/MM potential energy surfaces have been performed to study the D-glutamate --> L-glutamate reaction catalyzed by Bacillus subtilis glutamate racemase. The results show that the whole process involves four successive proton transfers that occur in three different steps. The Michaelis complex is already prepared to make the first proton transfer (from Cys74 to Asp10) possible. The second step involves two proton transfers (from the alpha-carbon to Cys74, and from Cys185 to the alpha-carbon), which occurs in a concerted way, although highly asynchronic. Finally, in the third step, the nascent deprotonated Cys185 is protonated by His187. The positively charged ammonium group of the substrate plays a very important key role in the reaction. It accompanies each proton transfer in a concerted and coupled way, but moving itself in the opposite direction from Asp10 to His187. Thus, the catalytic action of Bacillus subtilis glutamate racemase is driven by its own substrate of the reaction, D-glutamate.

Engineering the Promiscuous Racemase Activity of an Arylmalonate Decarboxylase

“…[7] Consequently, their molecular mechanism has received much attention in the last few years. Recent calculations for glutamate racemase (GluR) [8] and proline racemase (ProR) [7] suggest that the deprotonation and protonation is done simultaneously by two cysteines situated in an opposite orientation in the active site (Scheme 1 b, path b2). The introduction of a second Cys74 residue into arylmalonate decarboxylase (AMDase) from Bordatella bronchoseptica conferred a promiscuous racemising activity towards 2-arylaliphatic acids on it while the decarboxylating activity was retained.…”

“…[13] The residues of the hydrophobic pocket have a strong influence on the decarboxylating activity of the enzyme and were chosen as targets for a potential increase of the racemising activity. [14] For glutamate racemase (GluR), it has been suggested [8] that co-catalytic residues increase the nucleophilicity of the catalytic Cys residues by hydrogen bonding in order to facilitate the abstraction of the a proton. In AMDase G74C, the kinetic aproton acidity is increased by a tight hydrogen-bond network in the so-called dioxyanion hole.…”

Evaluation of new antimicrobial agents on Bacillus spp. strains: docking affinity and in vitro inhibition of glutamate-racemase

“…Therefore, the d-boroncontaining enantiomer must have this same structural geometry. The 11 B-NMR analysis showed a chemical shift of 4.5 ppm for both compounds, confirming the tetravalent structure 16,23 . Consequently, these two compounds can pass through the lipidic membrane of the bacteria relatively quickly, compared to tetracycline, in order to approach the active site.…”

“…Previous docking studies have used crystallized RacE in order to explore the active site of the RacE in detail 23 , identifying the amino acid residues that participate in the ligand-receptor interaction 3,8 . In the active site of RacE1, the natural substrate binds to Cys77 and Cys188, while in the active site of RacE2 this ligand binds to Cys74 and Cys185.…”

“…In the active site of RacE1, the natural substrate binds to Cys77 and Cys188, while in the active site of RacE2 this ligand binds to Cys74 and Cys185. Thus, residues change the configuration of glutamic acid from l to d or S to R by abstracting or adding a hydrogen atom 10,23 . In each of the enzyme isoforms evaluated, the three test compounds were relatively distant from the two principal residues involved in the catalytic activity by the natural substrates.…”