A single-residue mutation destabilizes Vibrio harveyi flavin reductase FRP dimer Navneet Jawanda a , Jerry Ebalunode a , Alexey Gribenko c , James Briggs a,b , J. Ching Lee c , Shiao-Chun Tu a,b, * 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 Received 15 November 2007, and in revised form 6 February 2008 Available online 12 February 2008 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 K d for the monomer–dimer equilibrium. The critical role of E99 in the formation of the FRP dimer has thus been demonstrated. Ó 2008 Published by Elsevier Inc. Keywords: Flavin reductase; Luciferase, bacterial; Vibrio harveyi; Subunit interaction; Dimmer dissociation; Monomer–dimer equilibrium With the exception of flavin reductase–luciferase couple from luminous bacteria, little was known about flavin- dependent two-component monooxygenase systems until the last decade, during which a growing number of such enzymes with diverse biological functions have been identi- fied and characterized [1,2]. Each two-component monoox- ygenase couple consists of a flavin reductase and a unifunctional monooxygenase. The latter relies on flavin reductase to supply reduced flavin as a substrate. Among all known flavin reductases, the NADPH-spe- cific flavin reductase FRP from Vibrio harveyi is one of the most extensively characterized. Our laboratory has a long term interest in the enzymology of FRP, both as an individual enzyme and with respect to the activity coupling with luciferase. One major line of our earlier and ongoing studies, including this work, focuses on the nature of FRP subunit interactions. Our crystallographic study reveals a homodimer structure of FRP with interlocking subunits, and provides a new structural fold to the flavoen- zymes family [3]. Each 26.3 kDa monomeric FRP contains one bound FMN cofactor [4]. FRP, for its size, has a remarkably large buried surface of 9352 A ˚ 2 and involves a multi-interaction network between the two subunits [3]. Yet, FRP readily undergoes in aqueous medium a rapid monomer–dimer equilibrium with a K d of 1.8 lM at 4 °C [5]. Moreover, the monomer–dimer equilibrium has great functional consequences. FRP has been shown to transfer its reduced flavin cofactor through direct channeling to luciferase within the FRP–luciferase complex for the cou- pled bioluminescence reaction [6–8]. However, V. harveyi luciferase forms a complex with the FRP monomer (K d , 0003-9861/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.abb.2008.02.006 * Corresponding author. Address: Department of Biology and Bio- chemistry, University of Houston, 4800 Calhoun, Houston, TX 77204- 5001, USA. Fax: +1 713 743 8351. E-mail address: dtu@uh.edu (S.-C. Tu). www.elsevier.com/locate/yabbi ABB Available online at www.sciencedirect.com Archives of Biochemistry and Biophysics 472 (2008) 51–57