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-directed mutagenesis of selected candidate residues of propanediol oxidoreductase from Escherichia coli. Based on the findings that mutations H263R, H267A and H277A resulted in iron-deficient propanediol oxidoreductases without catalytic activity, we identified three conserved His residues as iron ligands, which also bind zinc. The Cys362, a residue highly conserved among these dehydrogenases, was considered another possible ligand by comparison with the sequences of the medium-chain dehydrogenases. Mutation of Cys362 to Ile, resulted in an active enzyme that was still able to bind iron, with minor changes in the Km values and decreased thermal stability. Furthermore, in an attempt to produce an enzyme specific only for the zinc ion, three mutations were designed to mimic the catalytic zinc-binding site of the medium-chain dehydrogenases: (1) V262C produced an enzyme with altered kinetic parameters which 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 resulted in a catalytically inactive enzyme without iron-binding capacity, while retaining the ability to bind zinc. This mutation could represent a conceivable model of one of the steps in the evolution from iron to zinc-dependent dehydrogenases.
Obradors
N
N
Department de Bioquímica Facultat de Farmàcia, Universitat de Barcelona, Spain.
Cabiscol
E
E
Aguilar
J
J
Ros
J
J
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D017931:DNA Primers; D007501:Iron; D000429:Alcohol Oxidoreductases; C021494:lactaldehyde reductase