Crystal structures of vertebrate dihydropyrimidinase and complexes from Tetraodon nigroviridis with lysine carbamylation: metal and structural requirements for post-translational modification and function.

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala(69)-Arg(74) and Met(158)-Met(165), located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the monometal TnDhp, and in the "closed form" in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala(69)-Arg(74) dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-ß-alanine, and N-carbamyl-ß-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.

From sequence and structure of sulfotransferases and dihydropyrimidinases to an understanding of their mechanisms of action and function.

IMPORTANCE OF THE FIELD: Most enzymes that catalyze physiologically important reactions are known to be highly efficient and specific to their particular substrates. On the contrary, the enzymes of detoxification are known to catalyze unlimited number of substrates containing similar function groups that may have significant structural variation. This review explores nature's strategy to design enzymes with special properties. AREAS COVERED IN THIS REVIEW: We review articles from 1981 to 2009, with special focus on the relationships of sequence, structure and function of cytosolic sulfotransferases and dihydropyrimidinase (DHP). WHAT THE READER WILL GAIN: Specific amino acids responsible for substrate inhibition, substrate binding orientations, substrate specificity, quaternary structures and inactivation of sulfotransferases and DHP related enzymes are elucidated. Susceptibility to some diseases possibly resulted from the mutation of a single amino acid that causes dysfunction of these enzymes. Terminal deletion of amino acid that may affect surface interaction, subunit dissociation, stability alteration and then cause the syndrome of DHP deficiency is discussed. TAKE HOME MESSAGE: Based on the multiple sequence/structure analysis and with sufficient information from other members of the same enzyme families, the origin and mechanism of specific enzyme actions and proteins assembly can be clarified and predicted.

Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase.

Bacterial hydantoinase possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carboxylated lysine. How the carboxylated lysine and metal binding affect the activity of hydantoinase was investigated. A significant amount of iron was always found in Agrobacterium radiobacter hydantoinase purified from unsupplemented cobalt-, manganese-, or zinc-amended Escherichia coli cell cultures. A titration curve for the reactivation of apohydantoinase with cobalt indicates that the first metal was preferentially bound but did not give any enzyme activity until the second metal was also attached to the hydantoinase. The pH profiles of the metal-reconstituted hydantoinase were dependent on the specific metal ion bound to the active site, indicating a direct involvement of metal in catalysis. Mutation of the metal binding site residues, H57A, H59A, K148A, H181A, H237A, and D313A, completely abolished hydantoinase activity but preserved about half of the metal content, except for K148A, which lost both metals in its active site. However, the activity of K148A could be chemically rescued by short-chain carboxylic acids in the presence of cobalt, indicating that the carboxylated lysine was needed to coordinate the binuclear ion within the active site of hydantoinase. The mutant D313E enzyme was also active but resulted in a pH profile different from that of wild-type hydantoinase. A mechanism for hydantoinase involving metal, carboxylated K148, and D313 was proposed.