Entropic Stabilization of the Tryptophan Synthase α-Subunit from a Hyperthermophile, Pyrococcus furiosus

Abstract

The structure of the tryptophan synthase ␣-subunit from Pyrococcus furiosus was determined by x-ray analysis at 2.0-Å resolution, and its stability was examined by differential scanning calorimetry. Although the structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the ␣-subunit alone. The ␣-subunit from P. furiosus (Pf-␣-subunit) lacked 12 and 6 residues at the N and C termini, respectively, and one residue each in two loop regions as compared with that from S. typhimurium (St-␣-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix. The structure of the Pf-␣-subunit was essentially similar to that of the St-␣-subunit in the ␣ 2 ␤ 2 complex. The differences between both structures were discussed in connection with the higher stability of the Pf-␣-subunit and the complex formation of the ␣-and ␤-subunits. Calorimetric results indicated that the Pf-␣-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect. On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-␣-subunit. Rather, the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening of the polypeptide chain play important roles in extremely high stability in Pf-␣-subunit.Prokaryotic tryptophan synthase, which catalyzes the last processes in the biosynthesis of tryptophan, is a multienzyme ␣ 2 ␤ 2 complex composed of nonidentical ␣-and ␤-subunits. The separate ␣-and ␤ 2 -subunits catalyze inherent reactions termed ␣ and ␤ reactions, respectively. When the ␣-and ␤ 2 -subunits combine to form the ␣ 2 ␤ 2 complex, the enzymatic activity of each subunit is stimulated by 1 to 2 orders of magnitude (1). The ␣ 2 ␤ 2 complex has been studied as an excellent model system for seeking answers to important questions in proteinprotein interaction, especially in multifunctional enzymes. In 1988 (2) the three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium was determined by x-ray analysis. However, the structure of the ␣-or ␤ 2 -subunit alone has not yet been determined. To elucidate the molecular basis of the mutual activation of the subunit interaction due to the formation of the ␣ 2 ␤ 2 complex, we need to know the structures of the ␣-or ␤ 2 -subunits alone as well as that of the complex. Although the crystallization of each subunit from S. typhimurium and Escherichia coli has been tried for many years (3), the report of the x-ray structure has not yet appeared. Recently, the structures of a number of proteins from hyperthermophiles have been successfully determined by x-ray analysis. This seems due to the facts that proteins from hyperthermophiles are unusually stable and more easily form better crystals. Therefore, the ...

Stimulated Interaction between α and β Subunits of Tryptophan Synthase from Hyperthermophile Enhances Its Thermal Stability

“…The loops 2 and 6 in the St␣, which play an important role in the catalysis and allosteric communication between the active sites of the ␣ and ␤ subunits, contact with St␤ (23, 50 -52). The B-factor averaged for the main-chain atoms of the loop 2 in Pf␣ is considerably lower than that in St␣ (29), indicating that the loop 2 is less mobile in Pf␣ than in St␣. The loop 6 of St␣ is highly mobile and 12 residues in the loop 6 have not been determined due to a weak electron density (23).…”

“…Expression and Purification of ␣, ␤ 2 , and ␣ 2 ␤ 2 from P. furiosus-The ␣ subunit (Pf␣) from P. furiosus was expressed in the E. coli strain JM109/p␣1974 (30) and purified as described previously (29). Each of the genes of trpB and trpBA from P. furiosus was transformed into the E. coli strain JM109 (30).…”

“…24 -28). The thermal stability of the ␣ subunit of PfTSase is remarkably higher than that from Escherichia coli (29).…”

“…Structural Bases of Strong Subunit Association in PfTSaseThe structures of the St␣ 2 ␤ 2 complex form (23, 50 -52) and an isolated Pf␣ monomer (29) have been determined by x-ray analysis. We tried to explore the cause responsible for the strong subunit association in PfTSase from comparison of the structures of the subunit interfaces in Pf␣ and Ts␣.…”

“…␣ 2 ␤ 2 from P. furiosus-Pf␣, which consists of 248 residues and has a molecular weight of 27,500, is found to exist in a monomer form in solution (29). Ultracentrifugation analysis was used to determine the association forms of the proteins translated by the trpB and trpBA gens from P. furiosus, which were expressed in E. coli.…”