Crystal Structure of the βSer178 → Pro Mutant of Tryptophan Synthase

Abstract

The catalytic activity of the pyridoxal 5-phosphatedependent tryptophan synthase ␣ 2 ␤ 2 complex is allosterically regulated. The hydrogen bond between the helix ␤H6 residue ␤Ser 178 and the loop ␣L6 residue Gly 181 was shown to be critical in ligand-induced intersubunit signaling, with the ␣-␤ communication being completely lost in the mutant ␤Ser 178 3 Pro (Marabotti, A., De Biase, D., Tramonti, A., Bettati, S., and Mozzarelli, A. (2001) J. Biol. Chem. 276, 17747-17753). The structural basis of the impaired allosteric regulation was investigated by determining the crystal structures of the mutant ␤Ser 178 3 Pro in the absence and presence of the ␣-subunit ligands indole-3-acetylglycine and glycerol 3-phosphate. The mutation causes local and distant conformational changes especially in the ␤-subunit. The ligand-free structure exhibits larger differences at the N-terminal part of helix ␤H6, whereas the enzyme ligand complexes show differences at the C-terminal side. In contrast to the wild-type enzyme loop ␣L6 remains in an open conformation even in the presence of ␣-ligands. This effects the equilibrium between active and inactive conformations of the ␣-active site, altering k cat and K m , and forms the structural basis for the missing allosteric communication between the ␣-and ␤-subunits.The ␣ 2 ␤ 2 complex of tryptophan synthase (TRPS) 1 (EC 4.2.1.20) is a bifunctional enzyme that is considered a paradigm for the analysis of intersubunit regulatory signals. The enzyme is formed by two ␣-and ␤-subunits, arranged in a linear ␣␤␤␣ geometry (1), and catalyzes the last two steps in the biosynthesis of L-tryptophan in a highly concerted mode. The isolated ␣-and ␤-subunits exhibit an activity about 2 orders of magnitude lower that in the tetramer, indicating that the formation of the complex is accompanied by subunit conformational changes (2, 3). Further levels of regulation are achieved by ligand-induced intersubunit signals that are pH-, temperature-, and cation-dependent and keep the catalytic activities of ␣ and ␤-active sites in phase (4 -11). The mechanism of the ␣-␤ activation and allosteric regulation is based on an open-close transition of both subunits (7, 12, 13) involving the movement of several parts of the ␣-and ␤-subunits. In particular, signal transduction from the ␣-to the ␤-active site involves residues belonging to loop ␣L2, loop ␣L6, and the helix ␤H6 (14, 15), the latter structural element being part of the ␤Ϫsubunit COMM domain, as defined by Schneider et al. (15). The question whether there is a unique or multiple pathways of communication and whether these pathways are specialized in controlling defined functional properties of the enzyme was addressed by investigating the activity and regulatory properties of several mutants of the ␣-and ␤-subunits. It was found that mutants in loop ␣L2 and mutants in helix ␤H6 that interact with loop ␣L2 exhibit altered ␣ and ␤ activities (16 -18), whereas mutants in loop ␣L6 and mutants in helix ␤H6 interacting with loop ␣L6 also exhibit altered alloster...

pH Dependence of Tryptophan Synthase Catalytic Mechanism

“…The more relevant available structures are as follows: (i) the internal aldimine in the presence of either sodium (12,15,35,40), potassium, or cesium ions (40), also with bound ␣-subunit ligands (12,55); (ii) the external aldimine of the wild type enzyme in the presence of sodium ions (35); and (iii) the ␣-aminoacrylate Schiff base in the presence of sodium ions and 5-fluoroindole propanol phosphate, an ␣-subunit ligand (12). Other structures of the internal and external aldimine, determined on mutant enzymes (35,36,41,59,60), should be considered with caution, because mutation might have small but critical consequences on the location and local environment of ionizable residues. Furthermore, as pointed out earlier, no structure is available in the absence of monovalent cations at any stage of the catalytic cycle.…”

“…Surprisingly, no studies have addressed the pH dependence of the catalytic rates on the S. typhimurium enzyme, the form for which the three-dimensional structure of the internal aldimine (12,15,35), the external aldimine (35,36), and the ␣-aminoacrylate (12) has been determined. Recently, it was also found that monovalent cations and ␣-subunit ligands affect the distribution of intermediates as well as the catalytic properties of TS, triggering regulatory signals and stabilizing alternative open and closed conformations of the ␣-and ␤-subunits (23,27,(37)(38)(39)(40)(41). We have investigated the pH dependence of the catalytic rates in the absence and presence of monovalent cations and indole acetylglycine (IAG), a recently discovered ␣-subunit ligand (13,42).…”

Crystal Structures of a New Class of Allosteric Effectors Complexed to Tryptophan Synthase

“…The closed state of the ␣-subunit is communicated to the ␤-subunit via several interactions with the COMM domain, a rigid but moveable domain (␤Gly 102 -␤Gly 189 ) of the ␤-subunit (14). A critical element of the intersubunit interface is the hydrogen bond between ␣Gly 181 and ␤Ser 178 , as functionally demonstrated (22) and structurally characterized in the accompanying paper (23).…”

Identification of the Geometric Requirements for Allosteric Communication between the α- and β-Subunits of Tryptophan Synthase

“…The catalytic activity of ␣-and ␤-subunits is kept in phase by a fine-tuning associated with intersubunit communication (3,13,14). This allosteric regulation between active sites that are 20 Å apart (4,10) involves alternative open and closed states of both the ␣-and ␤-subunit (5,9,(15)(16)(17). The open to closed transition not only leads to an increase in the catalytic activity of the subunit but also is involved in signaling the catalytic state of one subunit to the other (3,13,14).…”

“…Indeed, the enzyme was still active but allosterically "knocked out." A structural study showed several alterations of the ␣-␤ interface and the inability of the ␣-subunit to achieve the closed state even in the presence of ␣-subunit ligands (17).…”