Chlorocatechol 1,2-dioxygenase from the Chlorophenol- utilizing Gram-positive Rhodococcus opacus 1CP: Crystallization and preliminary crystallographic analysis Maria Yolanda Ruiz Tarifa 1 , Marta Ferraroni 1 , Fabrizio Briganti 1 , Andrea Scozzafava 1 , Stefano Mangani 2 , Ludmilla Golovleva 1 Dipartimento di Chimica, Università di Firenze, Via Gino Capponi, 7 I-50121 Firenze, Italy 2 Dipartimento di Chimica, Università di Siena, Pian dei Mantellini, 44 I-53100 Siena, Italy 3 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142292 Pushchino Moscow region, Russia. The capability of Gram-positive bacteria to use chloroaromatics compounds as sole carbon and energy sources has been only recently observed. The majority of the representatives of the nocardiaform genus Rhodococcus do not show the presence of a modified ortho-cleavage pathway, first identified in Gram-negative strains for the assimilation of chloroaromatics. The aerobic metabolism of chloroaromatics, a class of compounds highly recalcitrant to biodegradation, generally occurs through two pathways depending on the number of chlorine substituents on the aromatic ring [1-2]. Those compounds having one or two chlorine are usually converted to chlorocatechols and then catabolized through the modified ortho-cleavage pathway, well studied in Gram-negative bacteria, whereas those containing two or more chlorine substituents are converted to hydroxyquinol or chlorohydroxyquinol [3-4]. Chlorocatechol 1,2-dioxygenases are key enzymes of the modified ortho-cleavage pathway and they generally show high substrate specificity. The chlorocatechol 1,2-dioxygenase (ClC1,2O) catalyzes the intradiol cleavage of chlorocatechols to chloro-cis,cis-muconates: OH OH COOH COOH O 2 Cl Cl Several ClC1,2Os have been purified from a variety of microorganisms but not much is known about the enzyme and the factors discriminating for substrate specificity of this novel group of intradiol dioxygenases. ClC1,2O from Rhodococcus opacus (erythropolis) 1CP is an homodimer of molecular weight of about 64 kDa, containing one Fe(III) and one Mn(II) ions [5]. A recent X-ray absorption spectroscopy study shows that, in the native enzyme, the iron is five-coordinated with average Fe-L distance of 1.93 Å and that histidines are present in the metal coordination sphere [6]. To date, only the X-ray structures of a few intradiol dioxygenases, the 3,4-protocatechuate dioxygenase (3,4PCD) from Pseudomonas aeruginosa and Acinetobacter Sp. ADP1, the Catechol 1,2-Dioxygenases from Acinetobacter Sp. ADP1 have been determined [7-8].