Multiple substrate binding states and chiral recognition in cofactor-independent glutamate racemase: a molecular...

Glutamate racemase (MurI) catalyzes the racemization of glutamate; two cysteine residues serve as catalytic acid and base. On the basis of the crystal structure of MurI from the hyperthermophilic bacterium Aquifex pyrophilus, we performed molecular dynamics (MD) simulations of six different systems to investigate stereochemistry, substrate ligation, and active site protonation state. The catalytic competence of individual systems was assessed by the abundance of reactive conformers. Only systems in which Cys70 is poised to deprotonate d-Glu were found to be catalytically competent (idem Cys178/l-Glu), in agreement with the experimentally observed stereochemistry of Lactobacillus fermentii MurI [Tanner, M. E. et al. (1993) Biochemistry 32, 3998-4006]. Only systems in which the alpha-amino group of l/d-Glu and the imidazole moiety of His are deprotonated are catalytically competent. The active site of MurI displays an unusual flexibility in substrate ligation, and several transitions between stable binding patterns were observed. In catalytically competent binding states, the conserved threonine residues 72, 114, and 117 ligate the alpha-carboxylate of Glu and the Asn71 amides ligate the alpha-amino group of Glu, whereas the delta-carboxylate of Glu is steered by electrostatic repulsion from the Asp7 and Glu147 side chain carboxylates. A network of hydrogen bonds controls the positioning of each thiol/thiolate. In what we term substrate flipping, Glu suddenly rotates into a binding pattern that resembles the post-racemization state of the other enantiomer, i.e., each enantiomer can be bound in two distinct states. Substrate flipping and unfavorable substrate binding successively trigger dissociation of the substrate, accompanied by an opening of the active site channel. We explain how the weak binding of Glu contributes to catalysis and suggest a mechanism by which binding mismatches are propagated into an opening of the active site.