Debbie S. Retnoningrum*, Hiromi Yoshida, Muthia D. Razani, Vincencius F. Meidianto, Andrian Hartanto, Anita Artarini and Wangsa T. Ismaya Pages 2 - 8 ( 7 )
Background: Bacterial manganese superoxide dismutase (MnSOD) occurs as a dimer, which is responsible for its activity and stability. Therefore, increasing the dimeric strength would increase the stability of the enzyme while maintaining its activity.
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 monomers.
Methods: 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 Xray crystallography.
Results: The N73F mutant activity was only ~40% of the wild type. The N73F mutant showed one TM at 60+1°C while the wild type has two (at 52-55°C and 63-67°C). 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, 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.
Dimeric strength, MnSOD, π-interaction, superoxide dismutation, Staphylococcus equorum, N73F.
Laboratory of Pharmaceutical Biotechnology, School of Pharmacy, Institut Teknologi Bandung, Bandung, Life Science Research Center and Faculty of Medicine, Kagawa University, Kagawa, Laboratory of Pharmaceutical Biotechnology, School of Pharmacy, Institut Teknologi Bandung, Bandung, Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, Laboratory of Pharmaceutical Biotechnology, School of Pharmacy, Institut Teknologi Bandung, Bandung, Laboratory of Pharmaceutical Biotechnology, School of Pharmacy, Institut Teknologi Bandung, Bandung, Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang