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 fromPyrococcus horikoshiiOT3 (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 groupP3212, with unit-cell parametersa=b= 57.2,c= 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å3 Da−1and 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.