title: Structural and biochemical properties of LuxF from Photobacterium leiognathi.
abstract: 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.
authors.affiliation: Graz University of Technology, Institute of Biochemistry, Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bergner
authors.affiliation: Graz University of Technology, Institute of Biochemistry, Graz, Austria.
authors.forename: Chaitanya R
authors.initials: CR
authors.lastname: Tabib
authors.affiliation: Graz University of Technology, Institute of Biochemistry, Graz, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Winkler
authors.affiliation: Graz University of Technology, Institute of Biochemistry, Graz, Austria.
authors.forename: Steve
authors.initials: S
authors.lastname: Stipsits
authors.affiliation: Graz University of Technology, Institute of Biochemistry, Graz, Austria.
authors.forename: Heidemarie
authors.initials: H
authors.lastname: Kayer
authors.affiliation: University of Georgia, Department of Biochemistry and Molecular Biology, Athens, GA 30602, USA.
authors.forename: John
authors.initials: J
authors.lastname: Lee
authors.affiliation: Conway Institute, University College Dublin, Dublin, Ireland.
authors.forename: J Paul
authors.initials: JP
authors.lastname: Malthouse
authors.affiliation: Conway Institute, University College Dublin, Dublin, Ireland.
authors.forename: Stephen
authors.initials: S
authors.lastname: Mayhew
authors.affiliation: Wylstrasse 13, CH-6052 Hergiswil (Formerly Novartis AG, Basel), Switzerland.
authors.forename: Franz
authors.initials: F
authors.lastname: Müller
authors.affiliation: University of Graz, Institute of Molecular Biosciences, Graz, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.affiliation: Graz University of Technology, Institute of Biochemistry, Graz, Austria. Electronic address: peter.macheroux@tugraz.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry*; D015870:Gene Expression; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049449:Luminescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000737:chemistry*; D010776:Photobacterium / Q000737:chemistry* / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019814:Myristic Acid; D005486:Flavin Mononucleotide; D008156:Luciferases
keywords: Bioluminescence; Isothermal calorimetry; Luciferase; Marine bacteria; UV–Vis absorption spectroscopy; X-ray crystallography