Bn archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Pseudomonas putida mt-2

Background: Catechol dioxygenases catalyze the ring cleavage of catechol an d its derivatives in either an intradiol or extradiol manner, These enzymes have a key role in the degradation of aromatic molecules in the environmen t by soil bacteria. Catechol 2,3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymu conate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively, The lack of an MPC struct ure has hampered the understanding of the general mechanism of extradiol di oxygenases. Results: The three-dimensional structure of MPC has been determined at 2.8 Angstrom resolution by the multiple isomorphous replacement method. The enz yme is a homotetramer with each subunit folded into two similar domains. Th e structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2- dioxygenase, although there is low amino acid sequence identity between the se enzymes. The active-site structure reveals a distorted tetrahedral Fe(II ) site with three endogenous ligands (His153, His214 and Glu265), and an ad ditional molecule that is most probably acetone. Conclusions: The present structure of MPG, combined with those of two 2,3-d ihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the a ctive site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The resu lts suggest that extradiol dioxygenases employ a common mechanism to recogn ize the catechol ring moiety of various substrates and to activate dioxygen . One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.