Preliminary crystallographic analysis of 3-chlorocatechol 1,2-dioxygenase of a new modified ortho-pathway from the Gram-positive Rhodococcus opacus 1CP grown on 2-chlorophenol.

3-Chlorocatechol 1,2-dioxygenase (3-ClC1,2DO), a key enzyme of a new modified ortho-pathway, was isolated from a variant of the Gram-positive bacterium Rhodococcus opacus 1CP utilizing 2-chlorophenol as the sole energy and carbon source via a 3-chlorocatechol branch of a modified ortho-pathway. 3-ClC1,2DO catalyzes the intradiol cleavage of 3-chlorocatechol. The enzyme contains Fe(III) ions essential to the catalytic activity; it is a homodimer with a molecular weight of about 58 kDa composed of two identical subunits in an (alphaFe)(2)-type quaternary structure. Its physicochemical properties are intermediate between those of the pyrocatechase from the ordinary pathway and those of the chloro-pyrocatechase from the modified pathway described previously for this strain. 3-ClC1,2DO was crystallized using the sitting-drop vapour-diffusion method. After 2 d, prismatic crystals grew in 15% PEG 8000, 0.3 M magnesium acetate, 100 mM HEPES pH 7.5, 5% glycerol. X-ray diffraction data were collected from a frozen crystal to a maximum resolution of 2.0 A using 25% PEG 400 as cryoprotectant at the Elettra synchrotron source, Trieste, Italy, at a wavelength of 1.01 A using a MAR CCD detector. The crystals belong to space group P1, with unit-cell parameters a = 83.18, b = 86.61, c = 93.44 A. Assuming a reasonable range for V(M), the asymmetric unit could contain from three to five (alphaFe(III))(2) dimers. A peak present in the kappa = 180 degrees and kappa = 90 degrees sections is consistent with a fourfold axis and four dimers in the asymmetric unit. Comparison of the crystal structure of this enzyme with that of the 4-chlorocatechol 1,2-dioxygenase recently crystallized from the same bacterium (Ferraroni et al., 2002) may reveal important details of the influence of the active-site conformation and the amino-acid substitutions involved in substrate selectivity.