title: The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional.
abstract: Human dihydrofolate reductase (DHFR) was previously thought to be the only enzyme capable of the reduction of dihydrofolate to tetrahydrofolate; an essential reaction necessary to ensure a continuous supply of biologically active folate. DHFR has been studied extensively from a number of perspectives because of its role in health and disease. Although the presence of a number of intronless DHFR pseudogenes has been known since the 1980s, it was assumed that none of these were expressed or functional. We show that humans do have a second dihydrofolate reductase enzyme encoded by the former pseudogene DHFRP4, located on chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate reductase-like 1 (DHFRL1), gene is expressed and shares some commonalities with DHFR. Recombinant DHFRL1 can complement a DHFR-negative phenotype in bacterial and mammalian cells but has a lower specific activity than DHFR. The K(m) for NADPH is similar for both enzymes but DHFRL1 has a higher K(m) for dihydrofolate when compared to DHFR. The need for a second reductase with lowered affinity for its substrate may fulfill a specific cellular requirement. The localization of DHFRL1 to the mitochondria, as demonstrated by confocal microscopy, indicates that mitochondrial dihydrofolate reductase activity may be optimal with a lowered affinity for dihydrofolate. We also found that DHFRL1 is capable of the same translational autoregulation as DHFR by binding to its own mRNA; with each enzyme also capable of replacing the other. The identification of DHFRL1 will have implications for previous research involving DHFR.
authors.affiliation: School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Gráinne
authors.initials: G
authors.lastname: McEntee
authors.forename: Stefano
authors.initials: S
authors.lastname: Minguzzi
authors.forename: Kirsty
authors.initials: K
authors.lastname: O'Brien
authors.forename: Nadia
authors.initials: N
authors.lastname: Ben Larbi
authors.forename: Christine
authors.initials: C
authors.lastname: Loscher
authors.forename: Ciarán
authors.initials: C
authors.lastname: O'Fágáin
authors.forename: Anne
authors.initials: A
authors.lastname: Parle-McDermott
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D005816:Genetic Complementation Test; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D058977:Molecular Sequence Annotation; D010641:Phenotype; D011485:Protein Binding; D011544:Pseudogenes / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase