Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.
A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.
Pettersson
Par L
PL
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden.
Johansson
Ann-Sofie
AS
Mannervik
Bengt
B
D001665:Binding Sites; D002384:Catalysis; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D010544:Peroxidases / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013251:Steroid Isomerases / Q000378:metabolism*
D011994:Recombinant Proteins; D010544:Peroxidases; D005982:Glutathione Transferase; D013251:Steroid Isomerases