Lys13 Plays a Crucial Role in the Functional Adaptation of the Thermophilic Triose-phosphate Isomerase fromBacillus stearothermophilus to High Temperatures

Abstract

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His 12 -Lys 13 pair instead of the Asn 12 -Gly 13 pair normally present in mesophilic TIMs. His 12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys 13 is unknown. To investigate the role of the His 12 and Lys 13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His 12 and Lys 13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-Å resolution. A molecular dynamics simulation showed that upon substitution of Lys 13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys 10 . This suggests that Lys 13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.Understanding the molecular adaptation of proteins to extreme temperatures remains a major challenge for protein scientists. Over the last 2 decades, several studies have attempted to establish a general rule leading to thermostability, but no overall consensus explanation could be formulated (1). For example, although the composition of thermophilic proteins shows that certain amino acids are preferred to ones present in mesophilic homologues, no general trends emerge. Studies have demonstrated the need to place the analysis of primary structure in a three-dimensional structural context (2); structural determinants such as ionic networks (3, 4), hydrophobic packing (5, 6), and cooperative associations (5, 7) seem to better reflect the general features associated with thermal adaptation. All of them, individually or in combination, enable thermophilic proteins to adapt to extreme temperatures.Thermotoga maritima is a hyperthermophilic eubacterium of the order Thermotogales, the oldest branch within the bacterial domain. Its optimal growth temperature is 80°C (8). Bacillus stearothermophilus is a moderately thermophilic endosporeforming Gram-positive rod growing optimally at about 65°C (9). Several glycolytic pathway enzymes from these strains have been produced by recombinant DNA technology, and all of them display optimal catalytic activity at temperatures above the optimal growth temperature of the source organism (1). Triose-phosphate isomerase (TIM) 1 is one of them. TIM is a homodimer catalyzing the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate in the glycolytic pathway (10). It is the prototype of a family comprising many different members sharing a common foldi...

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

“…The double mutation, H12N and K13G (His12 is mutated to an aspargine and a glycine residue replaces Lys13) affects residues present in loop 1, located at the dimer interface of the protein and seems to play a role in the thermostability of the protein (Alvarez et al, 1999).…”

“…Details of the crystallization and data collection have been presented elsewhere (Alvarez et al, 1999). The protein (7 mg ml À1 , 5 mM MES pH 6.5, 1 mM NaN 3 , 2 mM 2-phosphoglycolate) crystallizes against a 25%(w/v) PEG 4000 solution in 100 mM sodium acetate buffer pH 5.0 as a homodimer.…”

Structure and Function of a Regulated Archaeal Triosephosphate Isomerase Adapted to High Temperature

“…It has been characterised extensively both structurally and biochemically. Three-dimensional structures of TIMs from psychrophilic, 14 mesophilic 15-18 moderately thermophilic 19,20 and hyperthermophilic 1,4 organisms are known, thus allowing a comparative structural analysis across a broad temperature range of 15-100 8C.…”

Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins

“…S5, ESI ‡); 10,65,66 (ii) T m was independent of the scan rate (Fig. All these contrast with the thermal unfolding of NpTIM, GoTIM and all previously studied TIMs which are irreversible or present hysteresis 36,38,[43][44][45][46][47][48][49][50][51][52][53] and their high temperature CD spectra show the shape and a decrease in the spectral signal observed in unfolded and aggregated proteins (Fig. 4a-c) and (iv) catalytic activity of the refolded enzymes was between 84 and 96% of the native samples.…”

“…15,16 The Gnd-HCl and urea-induced unfolding of several monomeric TIM barrels has been studied in detail; [17][18][19] in contrast, there are only three reports on the reversible temperatureinduced unfolding of monomeric TIM barrels [20][21][22] and none describing an oligomeric TIM barrel. Even though full thermodynamic characterization has been hampered by irreversible aggregation and/or the presence of hysteresis in all of them, 36,38,[43][44][45][46][47][48][49][50][51][52][53] the activation parameters that describe the kinetic control of five eukaryotic TIMs have been reported. [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] Triosephosphate isomerase (E. C. 5.3.1.1) (TIM) is a key glycolytic enzyme that catalyzes the isomerization of glyceraldehyde-3phosphate (GAP) and dihydroxyacetone phosphate (DHAP).…”