Evolution of CuZn superoxide dismutase and the Greek Key β‐barrel structural motif

Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron‐exon boundary locations, gene duplication, and Greek key β‐barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of uperoxide dismutation. The β‐strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the β‐strands and at both termini. Thus, the β‐barrel with only four invariant residues is apparently over determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility ofthe Greek key β‐barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.