Fig 3.

Evidence that LeGCHI and AtGCHI encode functional enzymes. (A) Complementation of a yeast fol2 mutant. Similar numbers of cells of the wild-type 971/6c (WT), the fol2 deletant 971/6a (M), and 971/6a transformed with pVT103-U alone (V) or containing LeGCHI (Le) or AtGCHI (At) were plated on minimal medium plus or minus 50 μg⋅ml⁻¹ (6R,6S)-5-formyltetrahydrofolate. The minimal medium contained adenine, histidine, leucine, uracil, and tryptophan. Plates were incubated for 3 days at 30°C. (B) GCHI activities in desalted extracts of wild-type yeast strain 971/6a (WT), and the fol2 deletant harboring pVT103-U (V) alone or containing LeGCHI (Le), AtGCHI (At), or the N-terminal (Le-N) or C-terminal (Le-C) domain of LeGCHI. The data are means of three replicates ± SE. (C) Response of recombinant LeGCHI activity to GTP concentration. Assays contained 28 μg of yeast-extract protein and were incubated at 37°C for 1 h; product formation was linear during this time. Data points are means of 3–6 replicates ± SE. (D) Comigration in reverse-phase HPLC of the GCHI assay product with authentic neopterin. N, neopterin; WT, product of the wild-type yeast enzyme; Le, product of LeGCHI. (E) Comigration of authentic neopterin triphosphate (T) and of the oxidized but not phosphatase-treated product of partially purified LeGCHI (Le).