Crystallization and preliminary characterization of dihydropteridine reductase fromDictyostelium discoideum

Abstract

Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce d-threo-BH 4 [6R-(1 0 R,2 0 R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of l-erythro-BH 4 , in the last step of tetrahydrobiopterin (BH 4 ) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH 4 . To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR-NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 Â 0.6 Â 0.1 mm. The crystal belonged to space group P2 1 , with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 Å , = 100.00 , and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR-NAD dimers. Diffraction data were collected to 2.16 Å resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method.

Structural insights into the dual substrate specificities of mammalian andDictyosteliumdihydropteridine reductases toward two stereoisomers of quinonoid dihydrobiopterin

“…The purification, crystallization of the DicDHPR and the X-ray diffraction data collection were following the protocols described previously [17]. In brief, DicDHPR was purified by nickel-affinity, cation-exchange and size-exclusion chromatography.…”

“…The crystal structure of DicDHPR-NAD + was determined by the molecular replacement method as described before [17]. Then, several cycles of manual model building and refinement were performed using the program O [18] and CNS [19].…”