Ramelito C. Agapay, Savvas N. Savvides, Gonzalez Van Driessche, Bart Devreese, Jozef Van Beeumen, Jaap A. Jongejan, Wilfred R. Hagen
The RAM domain is one of several ligand-binding modules present in prokaryotes that are presumed to regulate the transcription of specific genes. To date, no hydrolytic activity has been reported for such modules. Curiously, a stand-alone RAM domain in Pyrococcus furiosus was isolated during a screen for hydrolytic activity against chromogenic esters. The gene encoding this protein was cloned and expressed in Escherichia coli and crystallized after a single purification step. X-ray diffraction data from the crystals were obtained to a resolution of 2.8 Å using a conventional X-ray source. The cocrystallization of the recombinant protein with 1,2-epoxy-3-(4-nitrophenoxy) propane (EPNP) and phenylmethylsulfonyl fluoride (PMSF) produced crystals that yielded data to 2.2 and 2.8 Å, respectively, using synchrotron radiation. Both the untreated and EPNP-treated crystals crystallize isomorphously in space group C2 and contain three dimers in the asymmetric unit. The PMSF-treated crystals also belong to this space group and have almost identical packing density, but show dramatically different unit-cell parameters. © 2005 International Union of Crystallography All rights reserved.
Department of Biotechnology, Delft University of Technology, 2628 BC Delft, Julianalaan 67, Netherlands; Chemical Engineering Department, University of San Carlos, Nasipit, Talamban, Cebu City 6000, Philippines; Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, 9000 Ghent, K. L. Ledeganckstraat 35, Belgium
