Background: Bacterial manganese superoxide dismutase (MnSOD) occurs as a dimer, which is responsible for
its activity and stability. Thereby, increasing the dimeric strength would increase the enzyme stability while maintaining its
Objective: An N73F substitution was introduced to strengthen interactions between the monomers at the dimer interface.
This substitution would introduce a π-stacking interaction between F73 of one monomer to F124 from the other monomer.
Method: Site-directed mutagenesis was carried out to substitute N73 with phenylalanine. The activity of the mutant was
qualitative- and quantitatively checked while the stability was evaluated with a fluorescence-based thermal-shift assay. Finally,
the structure of the mutant was elucidated by means of X-ray crystallography.
Results: The N73F mutant activity was only ~40% of the wildtype. The N73F mutant showed one TM at 60+1oC while the
wildtype has two (at 52-55oC and 63-67oC). The crystal structure of the mutant showed the interactions between F73 from
one monomer to F124 from the other monomer. The N73F structure presents an enigma because of no change in the enzyme
structure including the active site. Furthermore, N73 and F124 position and interaction are conserved in human MnSOD but
with a different location in the amino acid sequence. N73 has a role in the enzyme activity that is likely related to its interaction
with F124, which resides in the active site region but has not been considered to participate in the reaction.
Conclusion: The N73F substitution has revealed the unprecedented role of the N73-F124 pair in the enzyme activity.