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Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus. Kinetic and structural properties
M Alvarez1, J P Zeelen, V Mainfroid
1Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Sart Tilman, Belgium.
Abstract:
The purification and characterization of triose-phosphate isomerase from the psychrophilic bacterium Vibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-A resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15 degrees C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25 degrees C. The vTIM sequence is most closely related to the sequence of Escherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The Td values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54 degrees C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.
Insights
Researchers purified and characterized triose-phosphate isomerase (TIM) from a psychrophilic bacterium. A mutant protein showed increased stability, offering insights into enzyme engineering for cold environments.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Triose-phosphate isomerase (TIM) is a crucial enzyme in glycolysis.
- Psychrophilic enzymes offer unique insights into protein adaptation to cold temperatures.
- Vibrio marinus triose-phosphate isomerase (vTIM) is derived from a marine bacterium with optimal growth at 15°C.
Purpose of the Study:
- To purify and characterize vTIM.
- To determine the crystal structures of vTIM complexes.
- To investigate the structural and stability differences between vTIM and its mesophilic counterpart, Escherichia coli TIM (eTIM).
Main Methods:
- Protein purification and characterization.
- X-ray crystallography to determine protein structures.
- Differential scanning calorimetry (DSC) for thermal stability analysis.
- Site-directed mutagenesis to create the A238S mutant.
Main Results:
- vTIM was purified and characterized, revealing it to be unstable at temperatures above its optimal growth range (half-life of 10 min at 25°C).
- Crystal structures of vTIM-sulfate and vTIM-2-phosphoglycolate complexes were determined.
- Calorimetric studies showed vTIM has a lower melting temperature (Td = 41°C) compared to eTIM (Td = 54°C).
- A unique alanine at position 238 in loop 8 of vTIM was identified, differing from the conserved serine in other TIMs.
Conclusions:
- The instability of vTIM is linked to its unique structural features, particularly the alanine at position 238.
- Production and characterization of the vTIM mutant A238S demonstrated that substituting serine for alanine significantly increased protein stability.
- This suggests that specific amino acid substitutions can enhance the stability of psychrophilic enzymes, with potential applications in biotechnology.