Open AccessA preliminary crystallographic study of recombinant NicX, an Fe 2+ ‐dependent 2,5‐dihydroxypyridine dioxygenase from Pseudomonas putida KT2440
Author(s) -
Jiménez José Ignacio,
Acebrón Iván,
García José Luis,
Díaz Eduardo,
Mancheño José Miguel
Publication year - 2010
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430911001119x
Subject(s) - pseudomonas putida , dioxygenase , recombinant dna , materials science , crystallography , chemistry , enzyme , organic chemistry , biochemistry , gene
NicX from Pseudomonas putida KT2440 is an Fe 2+ ‐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 C 222 or C 222 1 . The estimated Matthews coefficient was 2.4 Å 3 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.