Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase fromPyrococcus horikoshiiOT3

Abstract

Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430-457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90-120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3(2)12, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å(3) Da(-1) and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution.

Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogenCryptococcus neoformans

“…AdSS from E. coli is arguably the best characterized (Silva et al, 1995;Poland et al, 1996;Choe et al, 1999) and has 42% identity to the cryptococcal enzyme. Crystal structures are also available for the proteins from Homo sapiens (54% identity; PDB entry 2v40; Structural Genomics Consortium, unpublished work), Mus musculus (54% identity; Iancu et al, 2001Iancu et al, , 2002, the bacteria Yersinia pestis (42% identity; PDB entry 3hid; R. Zhang, M. Zhou, S. Peterson, W. Anderson & A. Joachimiak, unpublished work), Campylobacter jejuni (43% identity; PDB entry 3r7t; Center for Structural Genomics of Infectious Diseases, unpublished work) and Burkholderia thailandensis (40% identity; PDB entry 3ue9; Seattle Structural Genomics Center for Infectious Disease, unpublished work), the plants Arabidopsis thaliana (55% identity) and Triticum aestivum (53% identity; Prade et al, 2000), the eukaryotic parasite Plasmodium falciparum (46% identity; Eaazhisai et al, 2004) and the extremophile archaeon Pyrococcus horikoshii (33% identity; Wang et al, 2011). The enzyme has been crystallized in the apo form (Silva et al, 1995;Iancu et al, 2001) as well as complexed with various combinations of IMP, GTP, GDP, aspartate, 6-PIMP, Mg 2+ and two AdSS inhibitors: the antitumour aspartate analogue hadacidin (Kaczka et al, 1962;Tibrewal & Elliott, 2011) and the adenosine 5 0 -monophosphatemimicking herbicide hydantocidin (Fonné -Pfister et al, 1996).…”

Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

“…Pyrococcus horikoshii (33%, (100) ). The enzyme has been crystallized in the apo-form (41,50) as well as complexed with various combinations of IMP, GTP, GDP, aspartate, 6-PIMP, Mg2+, and two AdSS inhibitors: the anti-tumor aspartate analogue hadacidin (56,61) and the adenosine 5'-monophosphate-mimicking herbicide hydantocidin (63) .…”