A preliminary crystallographic study of recombinant NicX, an Fe(2+)-dependent 2,5-dihydroxypyridine dioxygenase from Pseudomonas putida KT2440.
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 A at the ESRF. The crystals most probably belong to the orthorhombic space group C222 or C222(1). The estimated Matthews coefficient was 2.4 A(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.
Jiménez
José Ignacio
JI
Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Acebrón
Iván
I
García
José Luis
JL
Díaz
Eduardo
E
Mancheño
José Miguel
JM
D018360:Crystallography, X-Ray; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity
D006899:Mixed Function Oxygenases
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