title: 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.
abstract: 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.
authors.affiliation: Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (FI), Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Ferraroni
authors.forename: Maria Yolanda
authors.initials: MY
authors.lastname: Ruiz Tarifa
authors.forename: Andrea
authors.initials: A
authors.lastname: Scozzafava
authors.forename: Inna P
authors.initials: IP
authors.lastname: Solyanikova
authors.forename: Marina P
authors.initials: MP
authors.lastname: Kolomytseva
authors.forename: Ludmilla
authors.initials: L
authors.lastname: Golovleva
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Briganti
mesh_terms: D002733:Chlorophenols / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D049308:Dioxygenases; D001330:Electronic Data Processing; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology*; D012984:Software
chemical_list: D002733:Chlorophenols; D010105:Oxygenases; D049308:Dioxygenases; C044173:chlorocatechol 1,2-dioxygenase; C030683:2-chlorophenol