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

NicX 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 progress.