Conformational changes induced by cloxacillin in class a beta-lactamase from Bacillus cereus.

Class A beta-lactamases are enzymes that hydrolyse beta-lactam antibiotics such as penicillins and cephalosporins. They also hydrolyse substrate analogues such as oxacillin and cloxacillin, with a biphasic kinetic as it has been reported for Bacillus cereus beta-lactamase. A molecular model of Bacillus cereus beta-lactamase was built and the conformational changes that the substrates benzylpenicillin and cloxacilline produced in the conformation of selected regions of the protein were analyzed. This study was performed using the docking of the substrates, their tetrahedral intermediates and the corresponding acids on the active site, followed by molecular dynamic and subsequent optimisation procedures. The most important changes were produced on Tyr105 and Tyr273, when the tetrahedral intermediate of cloxacillin was docked at the active site, these amino acids are partially responsible for the stabilisation of the substrates at the active site. These changes may explain the kinetic differences observed during the hydrolysis of substrates type S and type A by beta-lactamases class A.

Crystallization and 2.2 A resolution structure of R-phycoerythrin from Gracilaria chilensis: a case of perfect hemihedral twinning.

R-phycoerythrin, a light-harvesting component from the red algae Gracilaria chilensis, was crystallized by vapour diffusion using ammonium sulfate as precipitant agent. Red crystals grew after one week at 293 K and diffracted to 2.70 A resolution. Three serial macroseeding assays were necessary to grow a second larger crystal to dimensions of 0.68 x 0.16 x 0.16 mm. This crystal diffracted to 2.24 A resolution using synchrotron radiation at beamline BM14 of the European Synchrotron Radiation Facility (ESRF) at Grenoble, France and was used for structure determination. Data were collected at 100 K to a completeness of 98.6%. The crystal was trigonal, space group R3, with unit-cell parameters a = b = 187.3, c = 59.1 A, alpha = beta = 90, gamma = 120 degrees. Data treatment using the CCP4 suite of programs indicated that the crystal was twinned ((I(2))/(I)(2) = 1.41). Molecular replacement was performed with AMoRe using the R-phycoerythrin from Polysiphonia urceolata [Chang et al. (1996), J. Mol. Biol. 249, 424-440] as a search model. In order to overcome the twinning problem, SHELX97 was used for the crystallographic refinement. The twin fraction was 0.48, indicating a nearly perfect hemihedrally twinned crystal. The final R(work) and R(free) factors are 0.16 and 0.25, respectively. All the residues and chromophores of the alpha- and beta-chains are well defined in the electron-density maps. Some residues belonging to the gamma-linker are also recognizable.

Ribonuclease T1 with free recognition and catalytic site: crystal structure analysis at 1.5 A resolution.

The free form of ribonuclease T1 (RNase T1) has been crystallized at neutral pH, and the three-dimensional structure of the enzyme has been determined at 1.5 A nominal resolution. Restrained least-squares refinement yielded an R value of 14.3% for 12,623 structure amplitudes. The high resolution of the structure analysis permits a detailed description of the solvent structure around RNase T1, the reliable rotational setting of several side-chain amide and imidazole groups and the identification of seven disordered residues. Among these, the disordered and completely internal Val78 residue is noteworthy. In the RNase T1 crystal structures determined thus far it is always disordered in the absence of bound guanosine, but not in its presence. A systematic analysis of hydrogen bonding reveals the presence in RNase T1 of 40 three-center and an additional seven four-center hydrogen bonds. Three-center hydrogen bonds occur predominantly in the alpha-helix, where their minor components close 3(10)-type turns, and in beta-sheets, where their minor components connect the peptide nitrogen and carbonyl functions of the same residue. The structure of the free form is compared with complexes of RNase T1 with filled base recognition site and/or catalytic site. Several structural rearrangements occurring upon inhibitor or substrate binding are clearly apparent. In conjunction with the available biochemical knowledge, they are used to describe probable steps occurring early during RNase T1-catalyzed phosphate transesterification.