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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 FeIII 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 (αFe)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 Å using 25% PEG 400 as cryoprotectant at the Elettra synchrotron source, Trieste, Italy, at a wavelength of 1.01 Å 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 Å. Assuming a reasonable range for VM, the asymmetric unit could contain from three to five (αFeIII)2 dimers. A peak present in the κ = 180° and κ = 90° 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.
