NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

Abstract

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography—the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry—to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4′ of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

NMR crystallography: structure and properties of materials from solid-state nuclear magnetic resonance observables

“…Another complex example is that of Caulkins et al (2016), who employed an integrated combination of multinuclear solid-state magnetic resonance, X-ray diffraction, and computational chemistry to characterize a carbanionic intermediate in tryptophan synthase. Their studies established a model of protonation states for ionizable groups on the cofactor, substrates, and proximal catalytic residues.…”

Engineered Biosynthesis of β‐Alkyl Tryptophan Analogues

“…Dehydration to form the electrophilic E(A-A) species occurs when TrpB populates a fully closed conformation, where it remains until product is formed. [28,29] To examine the state of the PfTrpB 7E6 active site and its connection to the COMM domain conformational cycling, we determined the X-ray crystal structures of PfTrpB 7E6 in the E(Ain) state as well as with β-EtSer bound in the active site as E(A-A). Earlier PfTrpB variants, including PfTrpB 2B9 , were nearly identical to wild-type PfTrpB (PDB: 5DVZ) in the open state.…”

Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

“…The molecular kinetics of the catalysis of tryptophan synthetase have been studied. The structure and reaction mechanism of tryptophan synthetase from Salmonella typhimurium have been analysed by NMR [23,24]. Excellent thermostability and catalytic activity of tryptophan synthase were needed in industrial production.…”