Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

Abstract

The tryptophan synthase α2β2 bi-enzyme complex catalyzes the last two steps in the synthesis of L-tryptophan (L-Trp). The α-subunit catalyzes cleavage of 3-indole-D-glycerol 3’-phosphate (IGP) to give indole and D-glyceraldehyde 3’-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25 Å-long tunnel, from the α-subunit to the (β-subunit where it reacts with L-Ser in a pyridoxal 5’-phosphate-dependent reaction to give L-Trp and a water molecule. The efficient utilization of IGP and L-Ser by tryptophan synthase to synthesize L-Trp utilizes a system of allosteric interactions that (1) function to switch the α-site on and off at different stages of the β-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the α- and β-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the α-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the bienzyme complex.

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

“…protein engineering | allostery | noncanonical amino acid | PLP H eteromeric enzyme complexes catalyzing a rich array of useful reactions are often allosterically regulated by their protein partners, such that the catalytic subunits are much less active when isolated (1)(2)(3). Utilization of isolated enzyme subunits, however, is desirable for biosynthetic applications, where expressing large complexes increases the metabolic load on the host cell and complicates efforts to engineer activity, substrate specificity, stability, and other properties.…”

“…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).…”

Tryptophan Synthase Uses an Atypical Mechanism To Achieve Substrate Specificity

“…In the absence of substrate Pf TrpS (20 μM) has an absorbance spectrum corresponding to E(Ain). 3 Addition of 20 mM L-Ser yields a spectrum with λ max = 350 nm. A similar peak with λ max = 345 nm accumulates when L-Thr is added.…”

“…1–3 The α-subunit (TrpA) catalyzes the retro-aldol cleavage of IGP, releasing indole into an intramolecular tunnel that forms when each subunit is in a closed conformational state and extends ~20 Å to the β-subunit (TrpB) active site. 4 TrpB utilizes a pyridoxal-phosphate (PLP) cofactor to effect the β-substitution of Ser with indole, yielding Trp.…”

“…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. 3 Detailed analysis of allosteric events awaits pre-steady state analysis; however, some reasonable hypotheses can be made drawing from the detailed literature of St TrpS. In the native catalytic cycle, IGP binding in the α-site stimulates E(A-A) formation by stabilizing the closed conformation of the COMM domain, which in turn signals for retro-aldol cleavage of IGP to release indole.…”