science • technique nr 12/2012 • tom 66 • 1349 Modern research methods for determining structures of intradiol dioxygenases Danuta WOJCIESZYŃSKA, Katarzyna HUPERT -KOCUREK, Małgorzata SITNIK, Urszula GUZIK – Department of Biotechnology, Faculty of Biology and Environmental Protection, University of Silesia in Katowice Please cite as: CHEMIK 2012, 66, 12, 1346-1351 Introduction In the era of a large interest in biodegradation processes of aromatic compounds, considerable attention has been directed to the studies on degrading enzymes, among which aromatic ring- cleavage dioxygenases have a key meaning [1,2]. Such enzymes catalyse the opening of a ring as a result of attaching two atoms of molecular oxygen and they are classified into two following groups depending on the regiospecificity of the ring cleavage: intradiol and extradiol [3]. The ring cleavage with the participation of intradiol dioxygenases does not only provide the degradation of uneasily degradable aromatic compounds such as: chlorophenols and nitrophenols, biphenyls and polycyclic aromatic hydrocarbons, but it also results in obtaining valuable intermediates for organic syntheses (cis, cis-muconic acid and 3-carboxy-cis, cis-muconic acid). Intradiol dioxygenases – enzymes involved in decomposition of aromatic compounds Intradiol dioxygenases catalyse the opening of the aromatic ring between two hydroxylated carbon atoms of the aromatic system, initiating the decomposition of aromatic compounds in the ortho pathway. It is a small family of enzymes with nonheme iron (III). They probably originate from the same evolutionary line. The analysis of spatial structure and amino acid sequence formed the basis for dividing dioxygenases from this group into the following: catechol 1,2-dioxygenases composed of α subunits, protocatechuate 3,4-dioxygenases composed of different numbers of αβ subunits and hydroxyquinol 1,2-dioxygenases composed of α subunits, similarly to catechol dioxygenases. Despite different subunit composition, catalytic domains of intradiol dioxygenases are formed similarly. In the active site of intradiol dioxygenases, there is iron in trigonal bipyramidal coordination geometry bound with four endogenous protein ligands. In the trigonal bipyramidal formation, the central atom is linked to 5 molecules at the tops of both pyramids [2]. Tyrosine 408, Histidine 460 and hydroxy group are attached to Fe (III) in the equatorial plane, whereas Tyrosine 447 and Histidine 462 in the axial plane. The composition of the active site is closely related to its function. The tests on the structure of enzyme-substrate complex of protocatechuate 3,4-dioxygenase showed that the attachment of substrate was associated with the separation of hydroxy group and Tyrosine 447, and the attached substrate gave its two protons to the detached ligands [4, 5]. Protocatechuate 3,4-dioxygenase is the enzyme that catalyses the transformation of protocatechuic acid into 3-carboxy-cis, cis-muconic acid [1,6,7]. This enzyme is characterised by the oligomer structure and is composed of two different types of subunits – α and β forming the (αβFe) n structure. β subunits combine protomers forming the oligomer structure and they are arranged along the axis of tetrahedron symmetry making the hollow sheath having the diameter of 50Å. α subunits are arranged in proximity to the peaks of two axes; they can be also found in the corners of opposite walls. Between α and β subunits, near the peak of the symmetry axis, there is an active site. The subunits are homologous towards each other. β subunit is composed of ca. 200 amino acid residues, while α subunit is composed of 230 amino acid residues. The secondary structure has the conformation of β-barrel constituted of β-sheet structure that is composed of 8 chains, twisted and coiled into the closed structure. It is similar to a piece of paper when folded in half [2, 5, 6]. Catechol 1,2-dioxygenases belong to the widely described class of enzymes [EC 1.13.11.1]. These enzymes cleave the catechol aromatic ring into cis,cis-muconic acid. Regarding the catalysed substrate, two subclasses of these enzymes are distinguished. They are: I – catechol dioxygenases catalysing the decomposition of catechol and methylcatechol, and less often chlorocatechol II – chlorocatechol dioxygenases decomposing catechol as well as its chlorinated and methylated forms [7÷10]. Catechol dioxygenases are homodimers of two identical α subunits, both of which contain Fe(III) cofactor. Each subunit is composed of ca. 300 amino acids and coiled into two domains: the catalytic domain whose structure is similar to the core of protocatechuate dioxygenases, and the terminal domain involved in dimerisation. N-terminal domain consists of ca. 100 amino acid residues making 5 helices. Dioxygenases of this class differ from other intradiol dioxygenases regarding both the subunit structure and the helical connector present at the subunit boundaries, to which phospholipid is being attached. The role of phospholipids has not been closely investigated so far [5, 6, 9]. Hydroxyquinol 1,2-dioxygenases belong to 3 rd group of intradiol dioxygenases [EC 1.13.11.37]. They catalyse hydroxyquinol transformation into 3-hydroxy-cis, cis-muconate. The model of spatial structure was described for hydroxyquinol dioxygenase of Nocardioides simplex strain 3E. It is a homodimer having the dimensions of 110x50x50 Å. Its structure and subunit composition (α 2 ) are similar to catechol dioxygenase, which is related to their close affinity. Despite that close affinity with catechol dioxygenases, hydroxyquinol dioxygenases have specific amino acid residues in their structure (Leu80, Asp83, Val107, Phe108, Gly109, Pro110, Phe111,Ile199, Pro200, Arg218, Val251) and big openings for attaching the substrate in the upper part of the active site [5, 8]. The intradiol mechanism of ring cleavage has not been fully investigated. The mechanism of intradiol aromatic ring cleavage was suggested on the basis of the analysis of the spatial structure of protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase and enzyme-substrate complexes as well as on the basis of biochemical and spectroscopic properties. The process of ring cleavage began from substrate attachment to the active site. The substrate gave two protons – one to hydroxy group, and the second to tyrosine in the axial position, which caused the deprotonation of both hydroxy groups of the substrate. Simultaneously, the dissociation of ligands coordinated with iron from the active site and the attachment of the dianonic substrate were observed. This led to the