UC Riverside

NMR-assisted crystallography is the integrated use of solid-state NMR, X-ray crystallography, and first-principles calculations to define the protonation states in an enzyme active site to give a chemically detailed three-dimensional structure. Determining hydrogen positions is a notable feature of the technique, complementing neutron diffraction but here NMR-assisted crystallography applies to catalytically active samples. This integrated approach was applied to the intermediates of Tryptophan synthase and Toho-1-β-lactamase. To determine an experimental structure, an unbiased set of protonation states are modeled and their predicted chemical shifts are compared to experimental shifts using the reduced-2 statistics. The confidence in the experimental structure can be quantified using Bayesian probability. From the structure the positional uncertainties are determined for the shift-rich region as well as the surrounding surface. NMR crystallography gives a remarkably clear picture of the chemistry of the β-subunit active site in tryptophan synthase. This level of detail reveals why BZI, an indole analog, does not proceed with the bond formation step despite being the better nucleophile: BZI is held in the wrong orientation by hydrogen bonds to the charged βLys87 and βGlu109 residues. This chemically-detailed view also reveals water positioned for nucleophilic attack on Cβ of the substrate. Based on its position and alignment, we posit that it sits in the initial binding pocket for the β-hydroxyl leaving group.