A preliminary crystallographic study of recombinant NicX, a Fe2+-dependent 2,5-dihydroxypyridine dioxygenase from Pseudomonas putida KT2440

5 páginas, 4 figuras, 1 tabla -- PAGS nros. 549-553NicX from Pseudomonas putida KT2440 is an Fe2+-dependent dioxygenase that is involved in the aerobic degradation of nicotinic acid. The enzyme converts 2,5-dihydroxypyridine to N-formylmaleamic acid when overexpressed in Escherichia coli. Biophysical characterization of NicX by analytical gel-filtration chromatography revealed that it behaves as an oligomeric assembly in solution, with an apparent molecular weight that is consistent with a hexameric species. NicX was crystallized by the hanging-drop vapour-diffusion method at 291 K. Diffraction data were collected to a resolution of 2.0 Å at the ESRF. The crystals most probably belong to the orthorhombic space group C222 or C2221. The estimated Matthews coefficient was 2.4 Å3 Da-1, corresponding to 50% solvent content, which is consistent with the presence of three protein molecules in the asymmetric unit. Analysis of the crystal data together with chromatographic results supports NicX being a hexameric assembly composed of two cyclic trimers. Currently, crystallization of recombinant selenomethionine-containing NicX is in progressJMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2007-67404/BMC), `Factoría de Cristalización' Consolider-Ingenio 2010 for support of his research and the ESRF (Grenoble, France) for provision of synchrotron-radiation facilities. ED and JLG acknowledge the Ministerio de Ciencia e Innovación for grants GEN2006-27750-C5-3-E, BIO2006-05957 and CSD2007-00005 and the Comunidad Autónoma de Madrid for grant P-AMB-259-0505. IA is the recipient of an FPU predoctoral fellowship from the Ministerio de Educación. JIJ was the recipient of an I3P predoctoral fellowship from the Consejo Superior de Investigaciones CientíficasPeer reviewe