Structural and biochemical properties of LuxF from Photobacterium leiognathi.

The lux-operon of bioluminescent bacteria contains the genes coding for the enzymes required for light emission. Some species of Photobacteria feature an additional gene, luxF, which shows similarity to luxA and luxB, the genes encoding the heterodimeric luciferase. Isolated dimeric LuxF binds four molecules of an unusually derivatized flavin, i.e., 6-(3'-(R)-myristyl)-FMN (myrFMN). In the present study we have heterologously expressed LuxF in Escherichia coli BL21 in order to advance our understanding of the protein's binding properties and its role in photobacterial bioluminescence. Structure determination by X-ray crystallography confirmed that apo-LuxF possesses four preorganized binding sites, which are further optimized by adjusting the orientation of amino acid side chains. To investigate the binding properties of recombinant LuxF we have isolated myrFMN from Photobacterium leiognathi S1. We found that LuxF binds myrFMN tightly with a dissociation constant of 80±20 nM demonstrating that the purified apo-form of LuxF is fully competent in myrFMN binding. In contrast to LuxF, binding of myrFMN to luciferase is much weaker (Kd=4.0±0.4 µM) enabling LuxF to prevent inhibition of the enzyme by scavenging myrFMN. Moreover, we have used apo-LuxF to demonstrate that myrFMN occurs in all Photobacteria tested, irrespective of the presence of luxF indicating that LuxF is not required for myrFMN biosynthesis.

Reverse structural genomics: an unusual flavin-binding site in a putative protease from Bacteroides thetaiotaomicron.

The structure of a putative protease from Bacteroides thetaiotaomicron features an unprecedented binding site for flavin mononucleotide. The flavin isoalloxazine ring is sandwiched between two tryptophan residues in the interface of the dimeric protein. We characterized the recombinant protein with regard to its affinity for naturally occurring flavin derivatives and several chemically modified flavin analogs. Dissociation constants were determined by isothermal titration calorimetry. The protein has high affinity to naturally occurring flavin derivatives, such as riboflavin, FMN, and FAD, as well as lumichrome, a photodegradation product of flavins. Similarly, chemically modified flavin analogs showed high affinity to the protein in the nanomolar range. Replacement of the tryptophan by phenylalanine gave rise to much weaker binding, whereas in the tryptophan to alanine variant, flavin binding was abolished. We propose that the protein is an unspecific scavenger of flavin compounds and may serve as a storage protein in vivo.