Flooding Enzymes: Quantifying the Contributions of Interstitial Water and Cavity Shape to Ligand Binding Using Extended Linear Response Free Energy Calculations

Glutamate racemase (GR) is a cofactor independent amino acid racemase that has recently garnered increasing attention as an antimicrobial drug target. There are numerous high resolution crystal structures of GR, yet these are invariably bound to either d-glutamate or very weakly bound oxygen-based salts. Recent in silico screens have identified a number of new competitive inhibitor scaffolds, which are not based on d-Glu, but exploit many of the same hydrogen bond donor positions. In silico studies on 1-<i>H</i>-benzimidazole-2-sulfonic acid (BISA) show that the sulfonic acid points to the back of the GR active site, in the most buried region, analogous to the C2-carboxylate binding position in the GR-d-glutamate complex. Furthermore, BISA has been shown to be the strongest nonamino acid competitive inhibitor. Previously published computational studies have suggested that a portion of this binding strength is derived from complexation with a more closed active site, relative to weaker ligands, and in which the internal water network is more isolated from the bulk solvent. In order to validate key contacts between the buried sulfonate moiety of BISA and moieties in the back of the enzyme active site, as well as to probe the energetic importance of the potentially large number of interstitial waters contacted by the BISA scaffold, we have designed several mutants of Asn75. GR-N75A removes a key hydrogen bond donor to the sulfonate of BISA, but also serves to introduce an additional interstitial water, due to the newly created space of the mutation. GR- N75L should also show the loss of a hydrogen bond donor to the sulfonate of BISA, but does not (a priori) seem to permit an additional interstitial water contact. In order to investigate the dynamics, structure, and energies of this water-mediated complexation, we have employed the extended linear response (ELR) approach for the calculation of binding free energies to GR, using the YASARA2 knowledge based force field on a set of ten GR complexes, and yielding an R-squared value of 0.85 and a RMSE of 2.0 kJ/mol. Surprisingly, the inhibitor set produces a uniformly large interstitial water contribution to the electrostatic interaction energy (⟨<i>V</i>^el⟩), ranging from 30 to >50%, except for the natural substrate (d-glutamate), which has only a 7% contribution of ⟨<i>V</i>^el⟩ from water. The broader implications for predicting and exploiting significant interstitial water contacts in ligand–enzyme complexation are discussed

Biosynthesis of a Novel Glutamate Racemase Containing a Site-Specific 7‑Hydroxycoumarin Amino Acid: Enzyme–Ligand Promiscuity Revealed at the Atomistic Level

Glutamate racemase (GR) catalyzes the cofactor independent stereoinversion of l- to d-glutamate for biosynthesis of bacterial cell walls. Because of its essential nature, this enzyme is under intense scrutiny as a drug target for the design of novel antimicrobial agents. However, the flexibility of the enzyme has made inhibitor design challenging. Previous steered molecular dynamics (MD), docking, and experimental studies have suggested that the enzyme forms highly varied complexes with different competitive inhibitor scaffolds. The current study employs a mutant orthogonal tRNA/aminoacyl-tRNA synthetase pair to genetically encode a non-natural fluorescent amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine (7HC), into a region (Tyr53) remote from the active site (previously identified by MD studies as undergoing ligand-associated changes) to generate an active mutant enzyme (GR^Y53/7HC). The GR^Y53/7HC enzyme is an active racemase, which permitted us to examine the nature of these idiosyncratic ligand-associated phenomena. One type of competitive inhibitor resulted in a dose-dependent quenching of the fluorescence of GR^Y53/7HC, while another type of competitive inhibitor resulted in a dose-dependent increase in fluorescence of GR^Y53/7HC. In order to investigate the environmental changes of the 7HC ring system that are distinctly associated with each of the GR^Y53/7HC–ligand complexes, and thus the source of the disparate quenching phenomena, a parallel computational study is described, which includes essential dynamics, ensemble docking and MD simulations of the relevant GR^Y53/7HC–ligand complexes. The changes in the solvent exposure of the 7HC ring system due to ligand-associated GR changes are consistent with the experimentally observed quenching phenomena. This study describes an approach for rationally predicting global protein allostery resulting from enzyme ligation to distinctive inhibitor scaffolds. The implications for fragment-based drug discovery and high throughput screening are discussed

Functional Comparison of the Two Bacillus anthracis Glutamate Racemases▿

Glutamate racemase activity in Bacillus anthracis is of significant interest with respect to chemotherapeutic drug design, because l-glutamate stereoisomerization to d-glutamate is predicted to be closely associated with peptidoglycan and capsule biosynthesis, which are important for growth and virulence, respectively. In contrast to most bacteria, which harbor a single glutamate racemase gene, the genomic sequence of B. anthracis predicts two genes encoding glutamate racemases, racE1 and racE2. To evaluate whether racE1 and racE2 encode functional glutamate racemases, we cloned and expressed racE1 and racE2 in Escherichia coli. Size exclusion chromatography of the two purified recombinant proteins suggested differences in their quaternary structures, as RacE1 eluted primarily as a monomer, while RacE2 demonstrated characteristics of a higher-order species. Analysis of purified recombinant RacE1 and RacE2 revealed that the two proteins catalyze the reversible stereoisomerization of l-glutamate and d-glutamate with similar, but not identical, steady-state kinetic properties. Analysis of the pH dependence of l-glutamate stereoisomerization suggested that RacE1 and RacE2 both possess two titratable active site residues important for catalysis. Moreover, directed mutagenesis of predicted active site residues resulted in complete attenuation of the enzymatic activities of both RacE1 and RacE2. Homology modeling of RacE1 and RacE2 revealed potential differences within the active site pocket that might affect the design of inhibitory pharmacophores. These results suggest that racE1 and racE2 encode functional glutamate racemases with similar, but not identical, active site features