Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3

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.

Figure 1

Alignment of the amino-acid sequences of EcAdSS, MiAdSS, PhAdSS and MjAdSS. Identical residues are shown as white letters on a red background, while similar residues are shown as red letters. The secondary-structural elements determined on the basis of the crystal structure of EcAdSS are indicated with arrows for β-strands and coils for α-­helices at the top of the alignment. The alignment was made using the program ClustalW (Larkin et al., 2007 ▶) and was coloured using the program ESpript (Gouet et al., 1999 ▶).

Figure 2

Typical crystal of PhAdSS with approximate dimensions of 0.15 × 0.1 × 0.1 mm grown by the microbatch-under-oil method.

Figure 3

Estimation of molecular weight in solution by native PAGE. Lane M contains molecular-weight standards (High Molecular Weight Calibration Kit for native electrophoresis from GE Healthcare). The molecular weight of each band is labelled in kDa on the left. The arrows indicate the two forms of recombinant PhAdSS with an N-terminal tag.