SITE-DIRECTED MUTAGENESIS STUDIES OF THE METAL-BINDING CENTER OF THE IRON-DEPENDENT PROPANEDIOL OXIDOREDUCTASE FROM ESCHERICHIA-COLI

The amino acid residues involved in the metal-binding site in the iron -containing dehydrogenase family were characterized by the site-direct ed mutagenesis of selected candidate residues of propanediol oxidoredu ctase from Escherichia coli. Based on the findings that mutations H263 R, H267A and H277A resulted in iron-deficient propanediol oxidoreducta ses without catalytic activity, we identified three conserved His resi dues as iron ligands, which also bind zinc. The Cys362, a residue high ly conserved among these dehydrogenases, was considered another possib le ligand by comparison with the sequences of the medium-chain dehydro genases. Mutation of Cys362 to ne, resulted in an active enzyme that w as still able to bind iron, with minor changes in the K-m values and d ecreased thermal stability. Furthermore, in an attempt to produce an e nzyme specific only for the zinc ion, three mutations were designed to mimic the catalytic zinc-binding site of the medium-chain dehydrogena ses: (1) V262C produced an enzyme with altered kinetic parameters whic h nevertheless retained a significant ability to bind both metals, (2) the double mutant V262C-M265D was inactive and too unstable to allow purification, and (3) the insertion of a cysteine at position 263 resu lted in a catalytically inactive enzyme without iron-binding capacity, while retaining the ability to bind zinc. This mutation could represe nt a conceivable model of one of the steps in the evolution from iron to zinc-dependent dehydrogenases.