title: Active site-directed double mutants of dihydrofolate reductase.
abstract: Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.
authors.affiliation: Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Ercikan-Abali
authors.forename: S
authors.initials: S
authors.lastname: Mineishi
authors.forename: Y
authors.initials: Y
authors.lastname: Tong
authors.forename: S
authors.initials: S
authors.lastname: Nakahara
authors.forename: M C
authors.initials: MC
authors.lastname: Waltham
authors.forename: D
authors.initials: D
authors.lastname: Banerjee
authors.forename: W
authors.initials: W
authors.lastname: Chen
authors.forename: M
authors.initials: M
authors.lastname: Sadelain
authors.forename: J R
authors.initials: JR
authors.lastname: Bertino
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000494:pharmacology*; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine
chemical_list: D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D013762:Tetrahydrofolate Dehydrogenase