NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the β-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the β-subunit from solution.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5′-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and β-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3′-phosphate and l-Ser to l-Trp. In this process, the roles played by β-site residues proximal to the PLP cofactor have not yet been fully established. βGln114 is one such residue. To explore the roles played by βQ114, we conducted a detailed investigation of the βQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet–visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the β-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and βQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of βGln114, βAsn145, and βArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (βT state) and closed (βR state) conformations of the β-subunit and consequently alter the distribution of intermediates along the β-subunit catalytic path.

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the 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. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

NMR-assisted crystallography – the synergistic combination 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 in the enzyme active site – including hydrogen atom locations and tautomeric equilibria – it offers insight into structure, dynamics, and function. Here, we make use of this combined approach to characterize the α-aminoacrylate intermediate in tryptophan synthase, a defining species for pyridoxal-5'-phosphate-dependent enzymes on the β-elimination and replacement pathway. By uniquely identifying the protonation states of ionizable sites on the cofactor, substrates, and catalytic side chains, as well as the location and orientation of structural waters in the active site, a remarkably clear picture of structure and reactivity emerges. Most incredibly, this intermediate appears to be mere tenths of angstroms away from the preceding transition state in which the β-hydroxyl of the serine substrate is lost. The position and orientation of the structural water immediately adjacent to the substrate β-carbon suggests not only the fate of the hydroxyl group, but also the pathway back to the transition state and the identity of the active site acid-base catalytic residue. Reaction of this intermediate with benzimidazole (BZI), an isostere of the natural substrate, indole, shows BZI bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the BZI position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack.

Solid-state NMR has emerged as a potent technique in structural biology, suitable for the study of fibrillar, micro-crystalline, and membrane proteins. Recent developments in fast-magic-angle-spinning and proton-detected methods have enabled detailed insights into structure and dynamics, but molecular-weight limitations for the asymmetric part of target proteins have remained at ~30-40 kDa. Here we employ solid-state NMR for atom-specific characterization of the 72 kDa (asymmetric unit) microcrystalline protein tryptophan synthase, an important target in pharmacology and biotechnology, chemical-shift assignments of which we obtain via higher-dimensionality, 4D and 5D solid-state NMR experiments. The assignments for the first time provide comprehensive data for assessment of side chain chemical properties involved in the catalytic turnover, and, in conjunction with first-principles calculations, precise determination of thermodynamic and kinetic parameters is demonstrated for the essential acid-base catalytic residue βK87. The insights provided by this study expand by nearly a factor of two the size limitations widely accepted for NMR today, demonstrating the applicability of solid-state NMR to systems that have been thought to be out of reach due to their complexity.