Analysis of metal-protein interaction distances, coordination numbers, B-factors (displacement parameters), and occupancies of metal binding sites in protein structures determined by X-ray crystallography and deposited in the PDB shows many unusual values and unexpected correlations. By measuring the frequency of each amino acid in metal ion binding sites, the positive or negative preferences of each residue for each type of cation were identified. Our approach may be used for fast identification of metal-binding structural motifs that cannot be identified on the basis of sequence similarity alone. The analysis compares data derived separately from high and medium resolution structures from the PDB with those from very high resolution small-molecule structures in the Cambridge Structural Database (CSD). For high resolution protein structures, the distribution of metal-protein or metal-water interaction distances agrees quite well with data from CSD, but the distribution is unrealistically wide for medium (2.0 -2.5 Å) resolution data. Our analysis of cation B-factors versus average B-factors of atoms in the cation environment reveals substantial numbers of structures contain either an incorrect metal ion assignment or an unusual coordination pattern. Correlation between data resolution and completeness of the metal coordination spheres is also found.

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...

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the shortchain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an Nterminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS⅐drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180°relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligandbinding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 Å) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH 2 H 4 folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure. Thymidylate synthase (TS)1 catalyzes the reductive methylation of 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) to thymidine 5Ј-monophosphate (dTMP), using the co-substrate, 5,10-methylenetetrahydrofolate (CH 2 H 4 folate) as a 1-carbon donor and reductant. The physical structures of bacterial TSs have been relatively well defined, and crystallographic data, in concert with data derived from kinetic, spectroscopic, and sitedirected mutagenesis studies, have led to a detailed understanding of the catalytic mechanism of these enzymes (1). In contrast, relatively few investigations of mammalian TS structure and catalysis have been conducted. The three-dimensional structure of the native human TS (hTS) has been reported previously (2). The data showed a surprising feature not observed in TSs from other sources: loop 181-197 containing the catalytic cysteine, Cys-195, was in an inactive conformation, rotated ϳ180°with respect to its orientation in bacterial TSs, with the sulfhydryl of Cys-195 over 10 Å from the location of sulfhydryls of corresponding cysteine residues in bacterial enzymes. Subsequent determination of the structure of a ternary inhibitory complex between closely related ratTS (rTS) and dUMP and Tomudex (3) has shown that the ligands bind to the enzyme in the active conformation. Recently, it was found that also in the hTS⅐dUMP⅐Tomudex complex hTS is in the active conformation (4). The inactive conformation has...

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