A single-residue mutation destabilizes Vibrio harveyi flavin reductase FRP dimer |
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Authors: | Jawanda Navneet Ebalunode Jerry Gribenko Alexey Briggs James Lee J Ching Tu Shiao-Chun |
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Affiliation: | a Department of Biology and Biochemistry, University of Houston, 4800 Calhoun, Houston, TX 77204-5001, USA b Department of Chemistry, University of Houston, Houston, TX 77204-5001, USA c Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX 77555-1055, USA |
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Abstract: | Our earlier studies have shown that the Vibrio harveyi flavin reductase FRP undergoes a monomer-dimer equilibrium, and luciferase forms a functional complex with the FRP monomer but not significantly with the dimer. This work is aimed at further investigating the nature and regulation of FRP subunit interactions by computation and site-directed mutagenesis approaches. In silico mutations of a number of residues were performed, and energetic analyses led us to target residue E99, which interacts directly with R113 and R225 from the second subunit of the FRP homodimer, for detailed investigation. E99 was found non-essential to the binding of either the FMN cofactor or the substrates. However, in comparison with the native enzyme, the E99K variant was shown to have an enhanced subunit dissociation as evident from a 44-fold higher Kd for the monomer-dimer equilibrium. The critical role of E99 in the formation of the FRP dimer has thus been demonstrated. |
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Keywords: | Flavin reductase Luciferase, bacterial Vibrio harveyi Subunit interaction Dimmer dissociation Monomer-dimer equilibrium |
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