Homo-dimeric recombinant dihydrofolate reductase from Thermotoga maritima shows extreme intrinsic stability.
Dihydrofolate reductase (DHFR) from the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. Sequence determination of the reported dyrA gene was repeated, and a corrected version deposited in the nucleotide sequence databank (accession number Y11021). Ultracentrifugational analysis and gel permeation chromatography prove that the enzyme forms a stable homodimer. The enzyme exhibits long-term stability at physiological temperature (80 degrees C) and in the presence of high denaturant concentrations (half-time in 6 M guanidinium chloride: 24h). Alignments of DHFRS from different species, as well as comparative modeling based on the homology to the crystal structures of the enzyme from prokaryotes and eukaryotes, were used to generate a model of the three-dimensional structure. The apoenzyme was crystallized and a data set was collected to a resolution of about 2 A.
Dams
T
T
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Böhm
G
G
Auerbach
G
G
Bader
G
G
Schurig
H
H
Jaenicke
R
R
D000595:Amino Acid Sequence; D019281:Dimerization; D004795:Enzyme Stability; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase