We have recently purified an intracellular carboxylesterase encoded by the open reading frame PA3859 of Pseudomonas aeruginosa. Among proteins showing a significant sequence homology with PA3859 the in vivo function is only known for the human acyl-protein thioesterase I that is involved in the deacylation of Galpha proteins. The crystal structure determination of P. aeruginosa carboxylesterase is expected to provide insights into its physiological role. Therefore, the PA3859 gene was cloned and heterologously expressed in Escherichia coli as N-terminally 6xHis tagged recombinant protein. Here, we present the crystallization, X-ray diffraction analysis and phasing of this enzyme. Two crystal forms were obtained by the hanging drop vapor diffusion method. Crystals of form I belong to the space group P2(1) with cell dimensions of a=65.65, b=50.55, c=142.55 A, beta=92.9 degrees and diffracted, upon flash annealing, up to a resolution of 2.9 A. Two dimers are present in the asymmetric unit. Crystals of form II belong to space group P2(1)2(1)2, with unit cell dimensions of a=96.42, b=96.36, c=68.04 A and diffracted up to 2.1 A resolution. One dimer is present in the asymmetric unit.
Crystallization, X-ray diffraction analysis and phasing of carboxylesterase PA3859 from Pseudomonas aeruginosa.
Pesaresi A;Lamba D
2005
Abstract
We have recently purified an intracellular carboxylesterase encoded by the open reading frame PA3859 of Pseudomonas aeruginosa. Among proteins showing a significant sequence homology with PA3859 the in vivo function is only known for the human acyl-protein thioesterase I that is involved in the deacylation of Galpha proteins. The crystal structure determination of P. aeruginosa carboxylesterase is expected to provide insights into its physiological role. Therefore, the PA3859 gene was cloned and heterologously expressed in Escherichia coli as N-terminally 6xHis tagged recombinant protein. Here, we present the crystallization, X-ray diffraction analysis and phasing of this enzyme. Two crystal forms were obtained by the hanging drop vapor diffusion method. Crystals of form I belong to the space group P2(1) with cell dimensions of a=65.65, b=50.55, c=142.55 A, beta=92.9 degrees and diffracted, upon flash annealing, up to a resolution of 2.9 A. Two dimers are present in the asymmetric unit. Crystals of form II belong to space group P2(1)2(1)2, with unit cell dimensions of a=96.42, b=96.36, c=68.04 A and diffracted up to 2.1 A resolution. One dimer is present in the asymmetric unit.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
