Tryptophan synthase: a mine for enzymologists

Abstract

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the last two steps of tryptophan biosynthesis in bacteria, plants and fungi. Structural, dynamic and functional studies, carried out over more than 40 years, have unveiled that: (1) alpha- and beta-active sites are separated by about 20 A and communicate via the selective stabilization of distinct conformational states, triggered by the chemical nature of individual catalytic intermediates and by allosteric ligands; (2) indole, formed at alpha-active site, is intramolecularly channeled to the beta-active site; and (3) naturally occurring as well as genetically generated mutants have allowed to pinpoint functional and regulatory roles for several individual amino acids. These key features have made tryptophan synthase a text-book case for the understanding of the interplay between chemistry and conformational energy landscapes.

Fig. 1

Structure of TS from Salmonella typhimurium. a Three-dimensional structure of TS α₂β₂ complex (pdb file 1K7E) [29]. The α-subunits are colored in pink and the β-subunits in green, with dark and light tones for the composing distinct domains. The α-active site is localized by the bound IAG, shown in blue sticks, and the β-active site by bound PLP, shown in red sticks. The monovalent cation bound in the β-subunit is shown as an orange sphere. The intramolecular channel connecting the α- and β-active sites is shown as a transparent volume only for a single α-β dimer. b Close-up view of the α-β subunit interface involved in the allosteric communication (pdb file 1K3U), showing interactions of α-loop6 and α-loop2 with β-helix6 of the COMM domain [13]. IAD bound at the α-active site is shown. Color code is the same as in panel a. c Open (red) and closed (pink) conformation of the α-subunit in the proximity of the subunit interface, associated to the allosteric regulation. The open conformation was obtained from molecular dynamics simulations [86] and the closed conformation from X-ray crystallography [29]

Fig. 2

Three-dimensional structure of the β-subunit active site of TS at different stage of catalysis. (a) Internal aldimine (pdb file 2clf [96]; (b) external aldimine with l-serine (pdb file 2clm [31]); (c) α-aminoacrylate in the presence of the α-subunit ligand α-d,l-glycerol-3-phosphate (pdb file 2j9x [31])

Scheme 1

Overview of the β-reaction catalyzed by tryptophan synthase

Scheme 2

Ketoenamine-enolimine equilibrium

Fig. 3

Effects of ligands and catalytic intermediates on the dominant conformation of the α- and β-subunits of TS. Abbreviations for catalytic intermediates of the β-reaction (see Scheme 1) are as follows: IA internal aldimine (open conformation), EA external aldimine (partially closed), AA α-aminoacrylate (closed), Q quinonoid species (closed). For simplicity, only some of the catalytic intermediates are shown. Dashed blue lines represent allosteric effects (allosteric ligands are framed in blue boxes). Substrates and products are framed in black boxes