Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus

Abstract

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.

FIG. 1.

Multiple alignment of amino acid sequences of selected reductases belonging to the SDR family, in order of similarity: D. radiodurans 3-oxoacyl-(acyl-carrier-protein) reductase (DrOAR), the homologous protein from E. coli (EcOAR), T. thermophilus DHPR (TtDH), DHPR 1 (nonquinonoid) from L. major (LmPTR1), quinonoid DHPR from L. major (LmqDPR), and rat DHPR. Bold type indicates identical residues in at least four of the six sequences. Sites conserved in the SDR family are underlined.

FIG. 2.

Partial purification of DH_Tt after cold precipitation, as shown by SDS-PAGE. Lane 1, molecular weight (MW) markers (in thousands, indicated on the left); lane 2, E. coli (pTTDH) clarified crude extract; lane 3, cold precipitate resuspended in 25 mM Tris-HCl (pH 10).

FIG. 3.

Activity staining of DH_Tt (A) and T. maritima DHFR (B) on electrophoresis gels. After electrophoresis of the proteins (see the text), gel pieces were either stained with Coomassie blue (lanes 1 and 2) or incubated as described in the text and as indicated above the lanes in the presence of substrates and/or cofactors and 5 mg of MMT/ml.