Ferraroni Marta, Ruiz Tarifa Maria Yolanda, Scozzafava Andrea, Solyanikova Inna P, Kolomytseva Marina P, Golovleva Ludmilla, Briganti Fabrizio
Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (FI), Italy.
Acta Crystallogr D Biol Crystallogr. 2003 Jan;59(Pt 1):188-90. doi: 10.1107/s0907444902020395. Epub 2002 Dec 19.
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.
3-氯儿茶酚1,2-双加氧酶（3-ClC1,2DO）是一种新的改良邻位途径的关键酶，它是从革兰氏阳性菌红平红球菌1CP的一个变种中分离得到的，该变种利用2-氯苯酚作为唯一的能量和碳源，通过改良邻位途径的3-氯儿茶酚分支进行代谢。3-ClC1,2DO催化3-氯儿茶酚的间位裂解。该酶含有对催化活性至关重要的Fe(III)离子；它是一种同型二聚体，分子量约为58 kDa，由两个相同的亚基组成，呈(αFe)(2)型四级结构。其物理化学性质介于普通途径的焦儿茶酚酶和该菌株先前描述的改良途径的氯焦儿茶酚酶之间。3-ClC1,2DO采用坐滴气相扩散法进行结晶。2天后，在含有15%聚乙二醇8000、0.3 M醋酸镁、100 mM HEPES（pH 7.5）、5%甘油的体系中长出棱柱形晶体。在意大利的里雅斯特的Elettra同步辐射源，使用MAR CCD探测器，以波长1.01 Å，将含有25%聚乙二醇400作为冷冻保护剂的冷冻晶体用于收集X射线衍射数据，最大分辨率达到2.0 Å。晶体属于空间群P1，晶胞参数a = 83.18、b = 86.61、c = 93.44 Å。假设V(M)在合理范围内，不对称单元可能包含三到五个(αFe(III))(2)二聚体。在κ = 180°和κ = 90°截面出现的一个峰与四重轴以及不对称单元中的四个二聚体一致。将该酶的晶体结构与最近从同一细菌中结晶得到的4-氯儿茶酚1,2-双加氧酶的晶体结构（Ferraroni等人，《生物化学杂志》，2002年）进行比较，可能会揭示活性位点构象和参与底物选择性的氨基酸取代影响的重要细节。
