From an inactive prokaryotic SOD homologue to an active protein through site-directed mutagenesis.
It is known that several prokaryotic protein sequences, characterized by high homology with the eukaryotic Cu,ZnSODs, lack some of the metal ligands. In the present work, we have stepwise reintroduced the two missing copper ligands in the SOD-like protein of Bacillus subtilis, through site-directed mutagenesis. The mutant with three out of the four His that bind copper is not active, whereas the fully reconstituted mutant displays an activity of about 10% that of human Cu,ZnSOD. The mutated proteins have been characterized in solution and in the solid state. In solution, the proteins experience conformational disorder, which is believed to be partly responsible for the decreased enzymatic activity and sheds light on the tendency of several human SOD mutants to introduce mobility in the protein frame. In the crystal, on the contrary, the protein has a well-defined conformation, giving rise to dimers through the coordination of an exogenous zinc ion. The catalytic properties of the double mutant, which might be regarded as a step in an artificial evolution from a nonactive SOD to a fully functioning enzyme, are discussed on the basis of the structural and dynamical properties.
Banci
Lucia
L
Department of Chemistry and Centro Risonanze Magnetiche, University of Florence, 50019 Sesto Fiorentino, Italy.
Benvenuti
Manuela
M
Bertini
Ivano
I
Cabelli
Diane E
DE
Calderone
Vito
V
Fantoni
Adele
A
Mangani
Stefano
S
Migliardi
Manuele
M
Viezzoli
Maria Silvia
MS
D001412:Bacillus subtilis / Q000737:chemistry; D003300:Copper / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011387:Prokaryotic Cells / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D008024:Ligands; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc