Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3.
The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 A. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V(M)) of 2.3 A(3) Da(-1), corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.
Manjunath
Kavyashree
K
Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560 012, India.
Jeyakanthan
Jeyaraman
J
Nakagawa
Noriko
N
Shinkai
Akeo
A
Yoshimura
Masato
M
Kuramitsu
Seiki
S
Yokoyama
Shigeyuki
S
Sekar
Kanagaraj
K
D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015870:Gene Expression; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D043902:Pyrococcus horikoshii / Q000201:enzymology*
D010453:Peptide Synthases; C022379:phosphoribosylaminoimidazole-succinocarboxamide synthetase