Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition.
Human manganese superoxide dismutase is a mitochondrial metalloenzyme that is involved in protecting aerobic organisms against superoxide toxicity, and has been implicated in slowing tumor growth. Unfortunately, this enzyme exhibits strong product inhibition, which limits its potential biomedical applications. Previous efforts to alleviate human manganese superoxide dismutase product inhibition utilized rational protein design and site-directed mutagenesis. These efforts led to variants of human manganese superoxide dismutase at residue 143 with dramatically reduced product inhibition, but also reduced catalytic activity and efficiency. Here, we report the use of a directed evolution approach to engineer two variants of the Q143A human manganese superoxide dismutase mutant enzyme with improved catalytic activity and efficiency. Two separate activity-restoring mutations were found--C140S and N73S--that increase the catalytic efficiency of the parent Q143A human manganese superoxide dismutase enzyme by up to five-fold while maintaining low product inhibition. Interestingly, C140S is a context-dependent mutation, and the C140S-Q143A human manganese superoxide dismutase did not follow Michaelis-Menten kinetics. The re-engineered human manganese superoxide dismutase mutants should be useful for biomedical applications, and our kinetic and structural studies also provide new insights into the structure-function relationships of human manganese superoxide dismutase.
Chockalingam
Karuppiah
K
Department of Chemical Engineering and Biomolecular Engineering, Institute for Genomic Biology, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Luba
James
J
Nick
Harry S
HS
Silverman
David N
DN
Zhao
Huimin
H
D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D013482:Superoxide Dismutase