Crystal structure of recombinant triosephosphate isomerase from bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three‐dimensional structures points to the importance of hydrophobic interactions

Abstract

The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the "closed" conformation in either subunit. The crystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 A for 248 C" atoms and have average temperature factors of 18.9 and 15.9 A*, respectively. In both subunits, the active site Lys 10 adopts an unusual $,$ combination.A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearotherrnophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearotherrnophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.

Identification of a potential metal cation–π binding site in the structure of a thermophilicBacillus stearothermophilustriosephosphate isomerase mutant

“…In fact, the cation lies in a position that is occupied by the lateral chain (CE/NZ) of Lys13 in the wild-type structure (Delboni et al, 1995;PDB code 1btm). Extensive general statistical analysis of side-chain interactions in well resolved protein structures showed that Tyr and Trp are over-represented as nearest neighbours of both Lys and Arg (Gallivan & Dougherty, 1999).…”

Phosphoenolpyruvate Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus

“…Unfortunately, whether S. marinus PEP synthetase is active has not been reported, as it was purified according to its size (8), and it is not known whether this organism contains a smaller (and active) version of the enzyme. It has been suggested that the increase in the number of subunits of hyperthermophilic enzymes compared to that for their mesophilic analogs might contribute to their increased thermostability (2,12,27,50). Whether the larger and seemingly inactive version of P. furiosus PEP synthetase is an artifact of purification or whether it has a physiological significance remains to be determined.…”

The tigA gene is a transcriptional fusion of glycolytic genes encoding triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in oomycota

“…The crystal structure of this enzyme has recently been determined from a bacterium (25) and a eukaryote (23). TPI is a homodimeric protein (EC 5.3.1.1), the crystal structure of which has been determined from a number of organisms (2,11,42). Comparative crystallographic studies have shown a high level of structural conservation between TPIs of diverse eukaryotic organisms (42).…”