Expression, purification and preliminary X-ray crystallographic analysis of nitroalkane oxidase (NAO) from Pseudomonas aeruginosa

Abstract

Nitroalkane oxidase (NAO) is a flavin-dependent enzyme which catalyses the oxidation of nitroalkanes to the corresponding aldehydes or ketones, nitrite and hydrogen peroxide. In order to better understand the structure and function of this enzyme, NAO from Pseudomonas aeruginosa was purified and crystallized as a native and a selenomethionine-substituted (SeMet) enzyme. Both crystals diffracted to a resolution of 1.9 angstrom and belonged to the primitive orthorhombic space group P2(1), with unit-cell parameters a = 70.06, b = 55.43, c = 87.74 angstrom, beta = 96.56 degrees for native NAO and a = 69.89, b = 54.83, c = 88.20 angstrom, beta = 95.79 degrees for SeMet NAO. Assuming the presence of two molecules in the asymmetric unit in both crystals, the Matthews coefficients (V-M) for native and SeMet NAO were calculated to be 2.30 and 2.48 angstrom(3) Da(-1), with estimated solvent contents of 46.50 and 50.37%, respectively.