Structural insights into the dual substrate specificities of mammalian and Dictyostelium dihydropteridine reductases toward two stereoisomers of quinonoid dihydrobiopterin

► The binary structure of DicDHPR-NAD + has been solved in 2.16 Å resolution. ► Structural changes in NADH binding site result in a higher K m for NADH than mammals. ► A mammalian DHPR carries a dual substrate activity verified by in vitro assay. ► We built structural models of the ratDHPR and DicDHPR in complex with two substrates. Up to now, d- threo-tetrahydrobiopterin (DH 4, dictyopterin) was detected only in Dictyostelium discoideum, while the isomer l- erythro-tetrahydrobioterin (BH 4) is common in mammals. To elucidate the mechanism of DH 4 regeneration by D. discoideum dihydropteridine reductase ( DicDHPR), we have determined the crystal structure of DicDHPR complexed with NAD + at 2.16 Å resolution. Significant structural differences from mammalian DHPRs are found around the coenzyme binding site, resulting in a higher K m value for NADH ( K m = 46.51 ± 0.4 μM) than mammals. In addition, we have found that rat DHPR as well as DicDHPR could bind to both substrates quinonoid-BH 2 and quinonoid-DH 2 by docking calculations and have confirmed their catalytic activity by in vitro assay. DHPR binds to DHPR by X-ray crystallography (View interaction)