Allostery and Substrate Channeling in the Tryptophan Synthase Bienzyme Complex: Evidence for Two Subunit Conformations and Four Quaternary States

Abstract

The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and β-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and β-catalytic cycles. 19F NMR studies of bound α-site substrate analogues, N-(4’-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4’-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of β-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of -0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α-β subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in 19F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and β-subunits have closed conformations wherein access of ligands into the α- and β-sites from solution is blocked. Internal and external aldimine structures show the α- and β-subunits with closed and open global conformations, respectively. These results establish that β-subunits exist in two global conformation states, designated open, where the β-sites are freely accessible to substrates, and closed, where the β-site portal into solution is blocked. Switching between these conformations is critically important for the αβ-catalytic cycle.

Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

“…1A) (2). The mechanism of this transformation has been extensively studied for TrpS from Escherichia coli and Salmonella typhimurium, where it has been shown the enzyme consists of two subunits, TrpA (α-subunit) and TrpB (β-subunit), both of which have low catalytic efficiencies in isolation (4). The activities of both subunits increase upon complex formation and are further regulated by an intricate and well-studied allosteric mechanism (2).…”

“…IGP binding to the α-subunit stimulates pyridoxal phosphate (PLP)-dependent aminoacrylate formation in the β-subunit [E(A-A); Fig. 1B], which in turn promotes retro-aldol cleavage of IGP in the α-subunit, releasing indole (4). This tightly choreographed mechanism serves to prevent the free diffusion of indole, which is only released from the α-subunit when the complex is in a closed conformation that forms a 25-Å tunnel through which indole diffuses into the β-subunit (5).…”

An Engineered Tryptophan Synthase Opens New Enzymatic Pathways to β‐Methyltryptophan and Derivatives

“…It is then suggested that βGlu109 deprotonates indole facilitating conjugate addition with (iv) leading to quinone (v) and L-Trp external aldimine (vi). (B) X-ray crystal structure (PDB4HPX) of the StTrpS β subunit [13] with an (E)-2-amino-2-butenoate intermediate ( In this paper we demonstrate how an engineered variant of tryptophan synthase (TrpS) can efficiently produce a range of enantiomerically pure (2S, 3S) L-β-methyltryptophan (β-mTrp) derivatives. Enzyme cascades utilizing the TrpS variant with Lamino acid oxidase (L-AAO) and halogenase enzymes provide access to more diverse L-and D-β-mTrp analogues.…”

“…Figure 1A). [15] Based on this information, combined with analysis of X-ray crystal structures of StTrpS [13] (Figure 1B), we predicted that the condensation of L-Thr and indole catalysed by StTrpS would proceed with indole addition to the Re face of the β-carbon in an (E)-2-amino-2-butenoate intermediate (iv, R = CH3) resulting in (2S,3S)-β-mTrp 1. As anticipated the configuration of the β-mTrp generated by both the wild-type StTrpS and the βL166V mutant was confirmed to be (2S,3S) by comparison of optical rotation and NMR data ( Figure S6), including a 3 JHαHβ coupling constant of 7.4 Hz, with data from literature.…”

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity

“…16 Hence, not only is the steady-state rate of catalysis lower with Thr, but the complex is likely in its closed state for less time. Notably, these open-close dynamics are an integral part of the TrpS allosteric signaling, 17 and altered TrpB conformational dynamics with Thr may disrupt the intricate reciprocal signaling between subunits that coordinates the catalytic cycle. These allosteric properties are sensitive to the identity of the monovalent cation bound to TrpS, and the present study focusses on the potassium form of the enzyme, as this is predominant intracellular monovalent cation.…”