Expression and site-directed mutagenesis of human dihydrofolate reductase

Abstract

A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-state kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6----Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the kcat for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the Km values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by alpha-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent Inhibitors of Cryptosporidium parvum Dihydrofolate Reductase: Structure-Activity and Structure-Selectivity Correlations

“…Briefly, all three proteins were expressed from dhfr mutant E. coli strain PA414 after transfection with the appropriate coding sequence and subcloning into expression plasmid pTrc99A, which contains a promoter induced with IPTG (isopropyl-␤-D-thiogalactopyranoside). In the case of the human enzyme, the E. coli mutant was transfected with previously described plasmid pDFR (50). All three enzymes were purified to homogeneity from their respective bacterial lysates by chromatography on a methotrexate-Sepharose affinity column.…”

“…Reactions were initiated by adding 100 l of 40 M dihydrofolate in assay buffer to an equal volume of assay buffer lacking dihydrofolate and containing various concentrations of the inhibitor. Each titration was performed twice, and the mean DHFR activity was plotted against the inhibitor concentration to obtain the IC 50 . Assays using E. coli DHFR were performed under the same conditions as those done with the human enzyme.…”

Efficacies of Lipophilic Inhibitors of Dihydrofolate Reductase against Parasitic Protozoa

“…The parent expression vector, pEH2, is derived from pRS314 (45,48). The DHFR-coding region from P. carinii (Pc-yeast) (11), S. cerevisiae (Sc-yeast) (12), or humans (Hu-yeast) (26) was cloned into the vector flanked at its 5Ј end by a portion of the yeast DHFR promoter region and at its 3Ј end by a portion of the yeast DHFR terminator, as described in detail by Brophy et al (4). In Apicomplexan parasites, the DHFR enzyme is one domain of a bifunctional protein that also contains the thymidylate synthase (TS) activity (5,18,46).…”

Antimycobacterial Activities of 2,4-Diamino-5-Deazapteridine Derivatives and Effects on Mycobacterial Dihydrofolate Reductase

“…Purified human rDHFR, which was generated using a recombinant system in Escherichia coli (23), was provided by Anatrace (Maumee, Ohio). According to the supplier, the enzyme has properties identical to those isolated from human cells as reported by Delcamp et al (8), and it contained an equimolar concentration of enzyme and dihydrofolate.…”