Genetic Engineering of a Type 2 DHFR
Abstract
Three areas of research on the Type II DHFR have been pursued. These were (1) analysis of structure and mechanism of the enzyme; (2) stability effects on the protein and variants in vivo and in vitro; and (3) plasmid constructs of mutants and expression of mutants. Studies of the first have shown that the stereochemistry of hydride transfer from NADPH to dihydrofolate placed this enzyme as one of the A-stereospecific class of dehydrogenases. The interaction of the enzyme with cofactors has been studied by equilibrium dialysis, proton NMR spectroscopy and differential scanning calorimetry. Two types of complexes have been found, and a shift of conformation between 5.0 and 5.9 was observed. In the mutant work, several mutants in the proposed active site have been formed. Some were inactive in vivo. In certain cases (e.g., Glu 58 yields Gln 58) the enzyme was rather unstable to protease attack in vivo. This factor was even more pronounced in sup + strains.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 11, 1991
- Accession Number
- ADA246249
Entities
People
- G. N. Bennett
Organizations
- Rice University