Structure of Sulfur-Substituted Rhodanese at 1.36 Å Resolution

Abstract

1.36 A Ê resolution X-ray diffraction data have been recorded at 100 K for bovine liver sulfur-substituted rhodanese, using synchrotron radiation. The crystal structure has been re®ned anisotropically to a ®nal R factor of 0.159 (R free = 0.229) for 53 034 unique re¯ections. The model contains 2 327 protein atoms and 407 solvent molecules, with a good geometry. The high resolution allows full details for helices, -sheets, tight turns and of all inter-and intramolecular interactions stabilizing the enzyme molecule to be given. The situation at the active site is described, particularly in regard to the network of hydrogen bonds made by S and S of the sulfur-substituted catalytic Cys247 and surrounding groups and solvent molecules. The replacement of the precipitant ammonium sulfate with cryoprotectants in the crystal-suspending medium led to the removal of the sulfate ion from the enzyme active site. Only limited changes of the enzyme structure have been found as a result of the drastic change in the crystal medium.

NH2-terminal Sequence Truncation Decreases the Stability of Bovine Rhodanese, Minimally Perturbs Its Crystal Structure, and Enhances Interaction with GroEL under Native Conditions

“…5), forming a network of hydrogen bonds among them and with protein atoms. A very similar situation is observed for the monoclinic crystal form transferred to a cryoprotectant solution devoid of ammonium sulfate (13): only one additional solvent molecule is visible at the active site, despite the significantly higher resolution of that data.…”

“…In a previous crystallographic study with monoclinic crystals, the presence of a sulfate ion in close proximity to the positive charges of Arg-186 and Lys-249 residues at the enzyme active site was demonstrated (27). When the mother liquid of the monoclinic crystals of rhodanese, containing ammonium sulfate, was exchanged with a cryo-protectant solution devoid of ammonium sulfate and containing PEG 6000 as precipitant (13), the density accounting for the sulfate ion at the active site was replaced by a peak of lower intensity, attributable to a water molecule. This observation is consistent with the finding of a substantial reactivation of the crystalline enzyme inhibited by sulfate, upon replacement of ammonium sulfate with PEG 6000 as precipitant in the crystal mother liquid.…”

“…Structure Solution and Refinement-The structure determination for wild type rhodanese in the orthorhombic crystal form was accomplished by means of the molecular replacement method, using the high resolution structure of sulfur-substituted rhodanese in the monoclinic crystal form as a starting model (13). The model, deprived of all solvent molecules, was positioned in a triclinic cell with 100-Å edges and a Patterson map was calculated.…”

“…Recently, the crystal structure has been refined to 1.36-Å resolution (13). The sulfur-free and sulfur-substituted enzymes crystallized isomorphously.…”

The Crystal Structure of Leishmania major 3-Mercaptopyruvate Sulfurtransferase

“…Crystal structures of rhodaneses have been elucidated and analyzed in detail (15)(16)(17)(18)(19)(20). The enzyme consists of two domains that, despite a low level of sequence identity, are structurally homologous.…”