Roles of divalent metal ions in flap endonuclease–substrate interactions

Abstract

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

“…This is presumably in part because a greater active site charge remains in the individual Ala mutants, but stabilizing substrate interactions with the side chains of Ile and Ser are also a possibility in D201I/D204S. A mutated T5FEN where the same two carboxylates are changed to arginine (D201R/D204R) retains activity in line with a non-catalytic substrate-positioning/stabilizing role for the T5FEN M2, perhaps compensating for the lack of a bound metal ion with the cationic arginine (12). The decrease in the reaction rate observed with our designed M2 mutant may reflect a change in the partitioning of the substrate structure between a bound duplex form and an unpaired, active site occupying form.…”

“…The N-terminal region of the protein is disordered in bacterial and bacteriophage FEN structures and is thought to be a site of protein-protein interaction (17,21,22). In T5FEN, the first 19 residues can be removed while retaining full enzymatic activity, and so the N terminus is unlikely to play a substrate-positioning role (12,23). Thus apparent discrepancies between metal ion-binding sites in FEN structures could be explained by a functional equivalence of a divalent metal ion and the N terminus.…”

“…analyses, including the observation that D201I/D204S mutations do not drastically impair reaction, suggest that the T5FEN M2 may not play a direct role in catalysis (12,24). In the designed D201I/D204S mutant, two of the carboxylates that bind M2 are altered (Fig.…”

“…Debate about the validity or otherwise of two-metal ion catalysis has focused on several enzymes including flap endonucleases (11)(12)(13)(14)(15)(16). Crystallographic studies of substrate-free FENs have demonstrated two active site-bound metal ions (14,(17)(18)(19).…”

MetalloPred: A tool for hierarchical prediction of metal ion binding proteins using cluster of neural networks and sequence derived features

“…The presence of the metal ion allows metalloenzymes to perform functions such as redox reactions that cannot be performed by the limited set of functional groups found in amino acids [1]. Metalloproteins play important roles in structural stability and complex formation [4][5][6][7][8], gene expression regulation and alteration [9][10][11][12], DNA processing [13], signaling processes and cellular event [14], transport [11,15,16], metabolism control [15,17], antibody recognition [18] and other biological processes such as cellular respiration, photosynthesis, nitrogen fixation and antioxidant defense [19]. Approximately, 1/3 of structurally-determined proteins are metalloproteins [20].…”