Crystallization of Chlorella deoxyuridine triphosphatase

Abstract

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous enzyme that has been widely studied owing to its function and evolutionary significance. The gene coding for the dUTPase from the Chlorella alga was codon-optimized and synthesized. The synthetic gene was expressed in Escherichia coli and recombinant core Chlorella dUTPase (chdUTPase) was purified. Crystallization of chdUTPase was performed by the repetitive hanging-drop vapor-diffusion method at 298 K with ammonium sulfate as the precipitant. In the presence of 2 0 -deoxyuridine-5 0 -[( , )-imido]triphosphate and magnesium, the enzyme produced die-shaped hexagonal R3 crystals with unit-cell parameters a = b = 66.9, c = 93.6 Å , = 120 . X-ray diffraction data for chdUTPase were collected to 1.6 Å resolution. The crystallization of chdUTPase with manganese resulted in very fragile clusters of needles.

Does a shift in host plants trigger speciation in the Alpine leaf beetle Oreina speciosissima(Coleoptera, Chrysomelidae)?

“…The genus Oreina currently includes twenty-eight species, of which only seven early-diverging taxa do not exclusively occur in high forbs (i.e. five develop in stone run vegetation and two can be found in both high forbs and stone runs) [ 32 ]. According to current knowledge [ 34 ], the most parsimonious explanation is that high forbs vegetation is the ancestral niche for the remaining twenty-one Oreina lineages, among which only our focal taxon Oreina speciosissima shows a partial reversal, since it is found both in high forbs and stone run vegetation.…”

“…Oreina speciosissima is distributed across nearly the entire range of the genus Oreina (from the Pyrenees in the west to the Carpathian Mountains in the east) through a wide altitudinal gradient (ranging from 800 to 2700 m above sea level). At lower elevations it generally colonizes the very abundant high forbs vegetation whereas at higher elevations it is found in stone run habitats across a small portion of its distribution range [unpublished observations MB, TVN][ 32 ]. Kippenberg [ 32 ] and personal observations suggest that Oreina speciosissima feeds exclusively on Asteraceae ( Achillea, Adenostyles, Cirsium, Doronicum, Petasites, Senecio and Tussilago ) and colonizes four distinct habitats represented by well-established plant associations: two occurring in high forbs - Petasition officinalis and Adenostylion - and two in stone run -Androsacion alpinae on siliceous bedrock and Petasition paradoxi on calcareous bedrock - (see Figure 1 )[ 33 ].…”

“…At lower elevations it generally colonizes the very abundant high forbs vegetation whereas at higher elevations it is found in stone run habitats across a small portion of its distribution range [unpublished observations MB, TVN][ 32 ]. Kippenberg [ 32 ] and personal observations suggest that Oreina speciosissima feeds exclusively on Asteraceae ( Achillea, Adenostyles, Cirsium, Doronicum, Petasites, Senecio and Tussilago ) and colonizes four distinct habitats represented by well-established plant associations: two occurring in high forbs - Petasition officinalis and Adenostylion - and two in stone run -Androsacion alpinae on siliceous bedrock and Petasition paradoxi on calcareous bedrock - (see Figure 1 )[ 33 ]. These plant communities are often patchily distributed due to the myriad of spatially proximate microclimates that occur in the Alps, especially sites with calcareous bedrocks which regularly present a mosaic of microhabitats.…”

Structural insights into the mechanism defining substrate affinity in Arabidopsis thaliana dUTPase: the role of tryptophan 93 in ligand orientation

“…Arabidopsis dUTPase was prepared as described previously [ 18 , 20 , 21 ]. Briefly, His-tagged Arabidopsis dUTPase was purified via Ni–NTA chromatography from the cleared lysate of E. coli JM103 (DE3) cells.…”

“…Crystals of holo Arabidopsis dUTPase were grown by vapor diffusion with the hanging drop method using 2 M ammonium sulfate as a precipitant in 50 mM Tris–HCl at pH 7.4 and 5 mM of the non-hydrolyzable ligand analog 2′-deoxyuridine 5′-[(α,β)-imido]-triphosphate (dUpNHpp; Jena Bioscience, Jena, Germany) along with 5 mM MgSO 4 (Table 2 ) [ 18 , 20 , 21 ].…”