Crystallization and X-ray analysis of theSchistosoma mansoniguanidino kinase

Abstract

The 716-amino-acid guanidino kinase from the parasitic flatworm Schistosoma mansoni results from the fusion of two guanidino kinase subunits. Crystals of this 80 kDa protein have been obtained in the monoclinic space group P2 1 , with unit-cell parameters a = 52.7, b = 122.1, c = 63.2 Å , = 108.5 . Synchrotron data were collected to 2.8 Å resolution on ESRF beamline ID29. The structure was solved by the molecular-replacement method, using the 357-amino-acid structure of the arginine kinase from Trypanosoma cruzi as the search model.

The Substrate-free and -bound Crystal Structures of the Duplicated Taurocyamine Kinase from the Human Parasite Schistosoma mansoni

“…Crystallization-We have previously published the crystallogenesis of SmTK and solved the phase problem by molecular replacement (38). However, the crystallographic refinement to 2.8 Å resolution led to the observation of a bound MES buffer molecule in the active site.…”

“…Construction, Overproduction, and Purification of EnzymesWild-type SmTK was overproduced and purified from Escherichia coli BL21(DE3) cells (Novagen) as previously reported (38). The invariant cysteine mutants C268S, C631S, and C268S/C631S (doubly mutated) were obtained by site-directed mutagenesis following the QuikChange site-directed mutagenesis protocol (Stratagene).…”

“…The XDS and XSCALE programs were used for data reduction and scaling (46). Five percent of the randomly selected reflections were kept apart for cross-validation, and the phasing problem was solved by molecular replacement as described previously (38). The substratefree structure was further refined by several cycles of rigid body in PHENIX (47) followed by positional and B-factor refinement.…”

“…A protein produced by this organism was classified as a TK (EC 2.7.3.4) based on sequence similarity and preliminary enzymatic assays (38). The S. mansoni TK (SmTK) is developmentally regulated and abundantly produced in the motile tail during the cercarial stage of the parasite's life cycle (39,40).…”

Phosphagen Kinase System of the Trematode Paragonimus westermani: Cloning and Expression of a Novel Chemotherapeutic Target

“…AKs were cloned from the nematodes Ascaris suum and Toxocara canis which can cause visceral larva migrans (VLM) in humans (Nagataki et al, 2008;Wickramasinghe et al, 2007) and from T. brucei which causes human sleeping sickness and Nagana in livestock (Pereira et al, 2002). The PK from the trematode Schistosoma mansoni was also recently described by Awama et al (2008). To determine the cDNA sequence of the PK in P. westermani, total RNA was first isolated from samples collected from definitive hosts in Bogil Island, South Korea using the acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski & Sacchi, 1987).…”