Conformational Changes in the Tryptophan Synthase from a Hyperthermophile upon α₂β₂ Complex Formation:  Crystal Structure of the Complex^,

Abstract

The three-dimensional structure of the bifunctional tryptophan synthase alpha(2)beta(2) complex from Pyrococcus furiosus was determined by crystallographic analysis. This crystal structure, with the structures of an alpha subunit monomer and a beta(2) subunit dimer that have already been reported, is the first structural set in which changes in structure that occur upon the association of the individual tryptophan synthase subunits were observed. To elucidate the structural basis of the stimulation of the enzymatic activity of each of the alpha and beta(2) subunits upon alpha(2)beta(2) complex formation, the conformational changes due to complex formation were analyzed in detail compared with the structures of the alpha monomer and beta(2) subunit dimer. The major conformational changes due to complex formation occurred in the region correlated with the catalytic function of the enzyme as follows. (1) Structural changes in the beta subunit were greater than those in the alpha subunit. (2) Large movements of A46 and L165 in the alpha subunit due to complex formation caused a more open conformation favoring the entry of the substrate at the alpha active site. (3) The major changes in the beta subunit were the broadening of a long tunnel through which the alpha subunit product (indole) is transferred to the beta active site and the opening of an entrance at the beta active site. (4) The changes in the conformations of both the alpha and beta subunits due to complex formation contributed to the stabilization of the subunit association, which is critical for the stimulation of the enzymatic activities.

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

“…We focused the search for an engineering starting point on known thermophilic TrpS enzymes for three reasons: (i) higher operating temperatures afford increased solubility of the hydrophobic substrates, which is useful for preparative reactions; (ii) thermostable enzymes are more tolerant to the introduction of activating but potentially destabilizing mutations (17); and (iii) thermostable enzyme variants can be screened efficiently (described below). We compared published kinetic properties of TrpS from Thermotoga maritima (18), Thermococcus kodakaraensis (19), and Pyrococcus furiosus (20)(21)(22) and selected the last for its superior kinetic parameters and thermostability (Table S1). In our hands, PfTrpB heterologously expressed and purified from E. coli has a k cat of 0.31 s −1 and experiences a 12-fold increase in catalytic efficiency upon addition of purified P. furiosus TrpA (PfTrpA) to make the PfTrpS complex (Table 1), similar to values reported previously for E. coli TrpB (EcTrpB) (15).…”

Modelling the evolution of the archeal tryptophan synthase

“…3D-data were deduced from the X-ray structure of Pfurio_i1E, i.e . the operon-based TrpB1 protein of P. furiosus [18], which has PDB code 1WDW. For Ssolfa_o2C, the 2D-structure was predicted by using Jpred [37].…”

“…This enzyme has been analysed for decades in order to understand the structural basis and functional consequences of protein-protein interactions [17]. The isolated TrpA and TrpB proteins form stable, however poorly active α monomers and ββ homodimers, respectively [18,19]. Their assembly to the native αββα complex induces conformational changes in both subunit types, as shown by X-ray crystallography for the Pyrococcus furiosus synthase [18].…”

“…The isolated TrpA and TrpB proteins form stable, however poorly active α monomers and ββ homodimers, respectively [18,19]. Their assembly to the native αββα complex induces conformational changes in both subunit types, as shown by X-ray crystallography for the Pyrococcus furiosus synthase [18]. The result of this communication between the α and β subunits is a reciprocal activation by one to two orders of magnitude [20].…”

Crystal Structure of a Homolog of Mammalian Serine Racemase from Schizosaccharomyces pombe

“…Mechanistic Implication of Wild-type Enzyme-The pyridine N-1 atom of the cofactor forms a hydrogen bond with the hydroxy group of Ser-308, acting as a hydrogen bond acceptor, as shown in the x-ray structures of threonine dehydratase (35), O-acetylserine sulfhydrylase (44,(51)(52)(53), threonine synthase (38,54,55), tryptophan synthase (40,56), and serine dehydratase (N-1-H-S hydrogen bond). The involvement of the N-1 atom in the neutral hydrogen bond with the hydroxy group of Ser or Thr or the sulfhydryl group of L-Cys as an acceptor is one of the characteristics of the fold-type II enzymes.…”