NMR-assisted crystallography – the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry – holds remarkable promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into structure and chemical dynamics. Here, we discuss the development and application of this combined approach to characterize several enzyme systems, including the pyridoxal-5'-phosphate dependent enzyme, tryptophan synthase (TS). For the TS α-aminoacrylate intermediate, NMR-assisted crystallography identifies the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains, and the location and orientation of crystallographic waters within the active site. From this, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the substrate L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate, indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react.
Enabling this approach is the ability to measure active-site isotropic and anisotropic NMR chemical shifts under conditions of active catalysis, and the development of fully quantum mechanical computational models of the enzyme active site that allow the accurate prediction of NMR spectral parameters. Overall confidence in the identification of the experimental structures is assigned using reduced-chi squared and Bayesian probability analysis, and positional uncertainties of the atom coordinates are quantitatively assessed.