The Crystal Structure and Amino Acid Sequence of Dehaloperoxidase from Amphitrite ornata Indicate Common Ancestry with Globins

Abstract

The full-length, protein coding sequence for dehaloperoxidase was obtained using a reverse genetic approach and a cDNA library from marine worm Amphitrite ornata. The crystal structure of the dehaloperoxidase (DHP) was determined by the multiple isomorphous replacement method and was refined at 1.8-Å resolution. The enzyme fold is that of the globin family and, together with the amino acid sequence information, indicates that the enzyme evolved from an ancient oxygen carrier. The peroxidase activity of DHP arose mainly through changes in the positions of the proximal and distal histidines relative to those seen in globins. The structure of a complex of DHP with 4-iodophenol is also reported, and it shows that in contrast to larger heme peroxidases DHP binds organic substrates in the distal cavity. The binding is facilitated by the histidine swinging in and out of the cavity. The modeled position of the oxygen atom bound to the heme suggests that the enzymatic reaction proceeds via direct attack of the oxygen atom on the carbon atom bound to the halogen atom.Polychlorinated phenols and other polychlorinated aromatics of anthropogenic origin have been widely dispersed and constitute significant environmental problems. It is less known that bromoaromatics of biotic origin are also widespread and secreted as chemical warfare by a number of marine organisms. Dehalogenating enzymes are used as the first line of defense against these toxicants by organisms that live in such contaminated environments (1). We have recently discovered and characterized by a number of techniques (2-4) an enzyme with a novel function, dehaloperoxidase (DHP).1 DHP is isolated from Amphitrite ornata, a terebellid polychaete. This species does not produce halogenated compounds itself but usually co-habits estuarine mud flats with other polychaete worms, such as Notomastus lobatus, and hemichordata such as Saccoglossus kowalewskyi, which secrete large quantities of brominated aromatics and other halometabolites as repellents (5). The levels of DHP are very high as it represents approximately 3% of the soluble protein in crude extracts of A. ornata. The enzyme catalyzes the oxidative dehalogenation of polyhalogenated phenols in the presence of hydrogen peroxide at a rate at least 10 times faster than all known halohydrolases of bacterial origin, according to Reaction 1.The oxidative potential of hydrogen peroxide likely allows for the unusually high rate of this reaction as well as for the unique ability of DHP to dehalogenate fluorophenols. The enzyme has activity toward substrates with different numbers and positions of halogen substituents (2).The binding of oxygen and peroxide ligands and their activation are due to the presence of heme in a variety of oxygen carriers and enzymes. This is also true for DHP, which contains one heme per subunit (3) and a histidine as the proximal iron ligand (4). The propensity of peroxidases (and oxygenases, which tend to have a cysteinate proximal ligand) to cleave the oxygen-oxygen bond and form a high vale...

Structure of dehaloperoxidase B at 1.58 Å resolution and structural characterization of the AB dimer fromAmphitrite ornata

“…Under the conditions employed, DHP B crystallized with two molecules in the asymmetric unit; the main-chain atoms of the two molecules superposed with an average displacement of 0.289 Å . These findings are analogous to those for DHP A, which also was found to crystallize as a homodimer (de Serrano et al, 2007;LaCount et al, 2000). Data-collection and refinement statistics are summarized in (Phillips, 2001) identified by the letters A-H (of which the segments Pro29-Asn34, Lys36-Tyr38 and Lys87-His89 assume 3 10 -helical conformations).…”

“…DHP is a bifunctional hemoprotein which acts as both the O 2 -transport protein and as a peroxidase in the terebellid polychaete Amphitrite ornata (LaCount et al, 2000;Chen et al, 1996), although how DHP performs these dual roles remains unclear at the present time. DHP has been shown to be a homodimer consisting of two identical subunits of $15.5 kDa in the asymmetric unit, each of which contains a heme protoporphyrin IX cofactor and eight -helices (de Serrano et al, 2007;Zhang et al, 1996).…”

“…These subunits are connected through an interface mainly comprised of salt bridges from acidic and basic amino-acid residues on the protein surface, specifically between Asp72 of one subunit and the side-chain groups of Arg122 and Asn126 of the other subunit (Lebioda et al, 1999;Chen et al, 2009). There is also a hydrophobic interaction between the Val74 residues of the two subunits (LaCount et al, 2000). The only cysteine present in DHP, Cys73, is located in close proximity to this interface.…”

“…To date, X-ray diffraction studies of dehaloperoxidase have been limited to one isoenzyme (DHP A; Chen et al, 2009; de Serrano et al, 2007;LaCount et al, 2000;Zhang et al, 1996;Lebioda et al, 1999). However, two isoforms of dehaloperoxidase, termed DHP A and DHP B, occur in A. ornata and are encoded by two separate genes (dhpA and dhpB; Han et al, 2001).…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…The active site of DHP consists of a protoporphyrin IX that is covalently attached to the apo-protein through the proximal His 89. In addition, the proximal His in DHP forms a hydrogen bond with Leu 83 that is weaker than that observed in other peroxidases and the distal pair of hydrophobic residues Arg-His, which is crucial for the enzymatic activity in other peroxidases, is missing in DHP (Franzen et al, 2006;LaCount et al, 2000;Osborne et al, 2004). The HemAT proteins belong to the globin coupled sensors family, which represent a unique class of oxygen sensing heme proteins found in both Archaea and Bacteria.…”