Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase.
We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.
Wilquet
V
V
Research Institute CERIA-COOVI, Brussels, Belgium.
Gaspar
J A
JA
van de Lande
M
M
Van de Casteele
M
M
Legrain
C
C
Meiering
E M
EM
Glansdorff
N
N
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008027:Light; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D012542:Scattering, Radiation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification*
D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase