title: Use of fortuitous in vitro mutations in a synthetic Cu-Zn superoxide dismutase gene to illuminate protein structure-function relationships.
abstract: A completely synthetic bovine copper-zinc superoxide dismutase gene (Cu-ZnSOD), designed using the most favoured codons for expression in yeast, was constructed. Fortuitous mutations introduced while cloning the synthetic gene permitted the additional construction of four altered-polypeptide products representing two single (Pro121-->Leu and Gly128-->Asp), one double (Pro100-->Leu, Arg113-->Lys) and one triple (Pro100-->Leu, Arg113-->Lys, Pro121-->Leu) mutant. All five versions of the gene were expressed in a SOD-deficient Escherichia coli strain. The 'wild-type' version of the gene and the two single-mutants were expressed to equal extents (approximately 8% of total soluble protein). However, compared with the 'wild-type' enzyme, one single-mutant (Gly128-->Asp) showed almost twice as much dismutase activity whilst the other (Pro121-->Leu) exhibited only 70% of the 'wild-type' level. In contrast, the double and triple mutants showed diminished expression of the gene (approximately 1 and 3% of total soluble protein, respectively) and almost no detectable SOD activity. Polyclonal antibovine SOD antibody bound all the recombinant proteins, although some of the products showed decreased size and probably altered conformations. The 'wild-type' superoxide dismutase recombinant was correctly dimerized and possessed dismutase activity, as did the Gly128-->Asp mutant despite the change in charge. Mutations in the other three versions affected enzyme folding and activity. The effect of the different mutations appeared to be additive, with the Pro121-->Leu substitution leading to the apparent proteolytic degradation of the enzyme in vivo.
authors.affiliation: Department of Biomolecular Sciences, UMIST, Manchester, UK.
authors.forename: J H
authors.initials: JH
authors.lastname: Kanan
authors.forename: S J
authors.initials: SJ
authors.lastname: Hubbard
authors.forename: S J
authors.initials: SJ
authors.lastname: Minter
authors.forename: S G
authors.initials: SG
authors.lastname: Oliver
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D013482:Superoxide Dismutase