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Dihydrofolate reductase (DHFR) enzyme catalyzes tetrahydrofolate regeneration by reduction of dihydrofolate using NADPH as a cofactor. Tetrahydrofolate and its one carbon adducts are required for de novo synthesis of purines and thymidylate, as well as glycine, methionine and serine. DHFR inhibition causes disruption of purine and thymidylate biosynthesis and DNA replication, leading to cell death. Therefore, DHFR has been an attractive target for chemotherapy of many diseases including cancer. Over the following years, in order to develop better antifolates, a detailed understanding of DHFR at every level has been undertaken such as structure-functional analysis, mechanisms of action, transcriptional and translation regulation of DHFR using a wide range of technologies. Because of this wealth of information created, DHFR has been used extensively as a model system for enzyme catalysis, investigating the relations between structure in-silico structure-based drug design, transcription from TATA-less promoters, regulation of transcription through the cell cycle, and translational autoregulation. In this review, the current understanding of human DHFR in terms of structure, function and regulation is summarized.

Dihydrofolate reductase (DHFR) enzyme catalyzes tetrahydrofolate regeneration by reduction of dihydrofolate using NADPH as a cofactor. Tetrahydrofolate and its one carbon adducts are required for de novo synthesis of purines and thymidylate, as well as glycine, methionine and serine. DHFR inhibition causes disruption of purine and thymidylate biosynthesis and DNA replication, leading to cell death. Therefore, DHFR has been an attractive target for chemotherapy of many diseases including cancer. Over the following years, in order to develop better antifolates, a detailed understanding of DHFR at every level has been undertaken such as structure-functional analysis, mechanisms of action, transcriptional and translation regulation of DHFR using a wide range of technologies. Because of this wealth of information created, DHFR has been used extensively as a model system for enzyme catalysis, investigating the relations between structure in-silico structure-based drug design, transcription from TATA-less promoters, regulation of transcription through the cell cycle, and translational autoregulation. In this review, the current understanding of human DHFR in terms of structure, function and regulation is summarized.

Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.