Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

Abstract

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.

Computational1H and13C NMR in structural and stereochemical studies

“…Indeed, very interesting results were reported very recently by Holmes, et al [ 390 ] on the imaging active site chemistry and protonation states, exemplified by the NMR crystallography of the tryptophan synthase α ‐aminoacrylate intermediate. In that paper, a combined approach based on a joint solid‐state NMR, X‐ray crystallography, and first‐principles computational approach, which enables the investigation of enzyme catalysis, was implied.…”

“… 15 N and 13 C CP MAS NMR spectra of microcrystalline TS, prepared with different isotopic labeling (see original paper for details). Reproduced from Holmes, et al [ 390 ] with the permission of the American Chemical Society under the Creative Commons Attribution‐NonCommercial 3.0 Unported Licence…”

“…Protonation states, hydrogen bonding interactions, and the placement and orientation of structural waters as revealed by NMR‐assisted crystallography in the TS β ‐subunit active site and related moieties (see original paper for details). Reproduced from Holmes, et al [ 390 ] with the permission of the American Chemical Society under the Creative Commons Attribution‐NonCommercial 3.0 Unported License…”

“…When combined with first‐principles computational chemistry, these complementary techniques can build consistent, testable models of structure and reactivity in enzyme active sites. Importantly, this can be accomplished for samples near room temperature and under conditions of active catalysis.” [ 390 ]…”

Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase

“…Recent publications include a variety of solution UV/Vis rapid kinetic studies (Harris et al, 2005;Ngo et al, 2007a, b;Dierkers et al, 2009;Ghosh et al, 2021;Phillips and Harris, 2021) and NMR studies employing ligands with 19 F (Niks et al, 2013) and 17 O probes (Young et al, 2016). Solid state magic angle spinning ssNMR investigations of TS complexes of substrate and substrate analogues and with 13 C and 15 N enriched PLP (McDowell et al, 1995;Lai et al, 2011;Caulkins et al, 2014Caulkins et al, , 2016Holmes et al, 2022), and β-subunits enriched with 15 N-Lys residues (Caulkins et al, 2014) and molecular dynamics studies (Huang et al, 2016;Maria-Solano et al, 2019;O'Rourke et al, 2019) have become especially important for establishing the predominant protonation states and tautomeric states of PLP intermediates in the β-reaction.…”