Cloning and overexpression of the triosephosphate isomerase genes from psychrophilic and thermophilic bacteria. Structural comparison of the predicted protein sequences.
We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues.
Rentier-Delrue
F
F
Laboratoire de Biologie Moléculaire, Université de Liège, Belgium.
Mande
S C
SC
Moyens
S
S
Terpstra
P
P
Mainfroid
V
V
Goraj
K
K
Lion
M
M
Hol
W G
WG
Martial
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008969:Molecular Sequence Data; D009016:Moraxella / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase