Protein Science zyxwvutsrqpo (1995), 4:2594-2604. Cambridge University Press. Printed in the USA Copyright zyxwvutsrqp 0 1995 The Protein Society Crystal structure of recombinant triosephosphate isomerase from Bacillus stearotherrnophilus. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions LUIS F. DELBONI,'.' SHEKHAR C. MANDE,' FRANCOISE RENTIER-DELRUE,3 VERONIQUE MAINFROID,3 STEWART TURLEY,' FREDERIQUE M.D. VELLIEUX,4 JOSEPH A. MARTIAL,3 zyxwvutsrq AND WIM G.J. HOL'.' zyxwvut ' Department of Biological Structure, Biomolecular Structure Center and *Howard Hughes Medical Institute, SL-15, School of Medicine, University of Washington, Seattle, Washington 98195 Laboratoire de Biologie Moleculaire et de Genie Genetique, Universite de Liege, B6, Sart Tilman, 4000 LIEGE, Belgium LCCP, CNRS 38027 Grenoble, Cedex 01, France (RECEIVED July 31, 1995; ACCEPTED October 5, 1995) 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 flat- tening was applied to improve the quality of the map. Active sites inboth the subunits are occupied by the inhibi- tor and the flexible loop adopts the "closed" conformation in either subunit. Thecrystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 zyxwvu A for 248 C" atoms andhave average tem- perature 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 stabil- ity of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stear- othermophilus 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 intro- ducing 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. Keywords: hydrophobicity; thermostability; triosephosphate isomerase " " - Triosephosphate isomerase (TIM) catalyzes the interconversion Reprint requests to: Wim G.J. Hol, Biomolecular Structure Center of ~-nlyceraldehyde-3-~hosphate (GAP) and dihydroxyacetone and the Howard Hughes Medical Institute, University of Washington, phosphate (DHAP) in the g~yco~ytic pathway. Only the dimer Box 357742, Seattle, Washington 98195; e-mail: sue@blackhook.bchem. washington.edu or zyxwvutsrq hol@gouda.bchem.washington.edu. is fully active (Waley, 1973; Zabori et al., 1980). Catalytic prop- zy s present address: Deoartmento de l+ica e ciencias dos Materias, erties of TIM have been studied in detail by numerous investi- " Universidade de Sao Paulb, Caix Postal 363, Sao Carlos, SP Brazil 13560. gators, employing kinetic experiments (Knowles & Albery, 2594