Construction of a catalytically active iron superoxide dismutase by rational protein design

Abstract

The rational protein design algorithm DEZYMER was used to introduce the active site of nonheme iron superoxide dismutase (SOD) into the hydrophobic interior of the host protein, Escherichia coli thioredoxin (Trx), a protein that does not naturally contain a transition metalbinding site. Reconstitution of the designed protein, Trx-SOD, showed the incorporation of one high-affinity metal-binding site. The electronic spectra of the holoprotein and its N 3 ؊ and F ؊ adducts are analogous to those previously reported for native {Fe 3؉ }SOD. Activity assays showed that {Fe 3؉ }Trx-SOD is capable of catalyzing the dismutation of the superoxide anion; comparative studies with the unrelated wild-type E. coli iron SOD indicated that {Fe 3؉ }Trx-SOD catalyzes the dismutation reaction at a rate on the order of 10 5 M ؊1 s ؊1 . The ability to design catalytically competent metalloenzymes allows for the systematic investigation of fundamental mechanistic questions concerning catalysis at transition metal centers.Protein design methodologies (1-3) have recently focused on incorporating transition metal ions into proteins (3-6) to take advantage of their well-documented catalytic or electron transfer functions. However, this long-term objective of rationally engineering metalloproteins to introduce novel catalytic functions has heretofore met with only limited success (5-8). We have used the rational protein design algorithm DEZYMER (9) to test our ability to design catalytically active metalloenzymes by constructing a five-coordinate nonheme iron active site analogous to that found in iron-dependent superoxide dismutase (SOD) (10, 11) in a host protein, thioredoxin (Trx) (Fig. 1). The construction of this site represents a significantly different challenge from the design of coordinatively saturated, enzymatically inert, metal sites (6,8,40) by attempting to build a site that catalyzes the inner sphere transfer of electrons both to and from small molecule substrates. For this chemistry to occur, the availability of an open coordination position, or one occupied by a labile ligand such as H 2 O, is required, but the site must be sufficiently buried to prevent protein dimerization via a metal bridge. Introduction of a cavity for anion binding, as well as other factors relating to the attraction and transport of the anion to the active site, need to be considered as well.The DEZYMER algorithm makes predictions based on strict geometric principles without explicit consideration of binding thermodynamics or protein dynamics. The rational design approach used here is based on the placement of an active site into the framework of a known protein fold. Active sites are described as geometrical arrangements of functionally important amino acids around a ligand, in this case a transition metal center. In the first phase of the search, the DEZYMER algorithm systematically examines a protein structure to identify backbone positions that are arranged in such a way that appropriate rotamers of the residues in the binding-site defini...

Enzyme-like proteins by computational design

“…Computational techniques have been used to design novel metal binding sites into proteins (11)(12)(13). By leaving one of the primary coordination spheres of the metal unligated by the protein, nascent metalloenzymes with a variety of oxygen redox chemistries have been generated (14,15). The general design of metalloenzymes to react specifically with more complicated organic molecules has not been demonstrated.…”

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

“…This problem may be circumvented by grafting inorganic cofactor-binding sites into the structures of natural proteins that normally do not bind metal ions. Automated methods have been developed recently for engineering such ion-binding sites (15, 16), and it has been possible to build a number of structural as well as redox-active metal ion-binding sites within several different proteins (17)(18)(19)(20). Another successful approach has been to design small flexible peptides that are able to fold around metal sites such as Cu(II)-binding motifs (21-24) and small peptides that assemble into Fe 4 S 4 clusters (25,26).…”

Changes in activity during storage and characteristics of superoxide dismutase from hen eggs (Gallus gallus domesticus)

“…3) was shown, which confirmed the presence of Mn ions in enzymatic protein, but not confirmed by electrophoresis (Fig. 4) [32][33][34].…”