Crystallographic Comparison of Manganese- and Iron-Dependent Homoprotocatechuate 2,3-Dioxygenases

Abstract

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn 2؉ and Fe 2؉ , respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four ␤␣␤␤␤ modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214 NE1 and E267 OE1 ; one axial ligand, H155 NE1 ; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 Å), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O 2 . The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O 2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.Bacterial ring-cleaving dioxygenases are critical enzymes in the catabolism of aromatic compounds that enter the environment from a myriad of sources (9,17,18,44). The substrates for most of these enzymes are ortho-or para-dihydroxylated aromatics and molecular oxygen. Both atoms of oxygen from O 2 are incorporated into the product as the ring is cleaved. The resulting aliphatic products are further metabolized to intermediates of core metabolic cycles through well-established pathways (18,55,74).Dioxygenases that act on ortho-dihydroxylated aromatic compounds are divided into two classes, termed intradiol and extradiol, which differ in their mode of ring cleavage and the oxidation state of the active-site metal (44, 63). Intradiol dioxygenases utilize Fe 3ϩ and cleave the bond between the carbons bearing the two hydroxyls; extradiol dioxygenases utilize Fe 2ϩ or, rarely, Mn 2ϩ and cleave one of the carbon-carbon bonds adjacent to the ortho-hydroxyl substituents. Although intradiol and extradiol dioxygenases oxidize an overlapping set of substrates, they are not structurally related and are proposed to have fundamentally differ...

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