Crystal Structures of the Lumazine Protein from Photobacterium kishitanii in Complexes with the Authentic Chromophore, 6,7-Dimethyl- 8-(1′-d-Ribityl) Lumazine,and Its Analogues,Riboflavin and Flavin Mononucleotide,at High Resolution

Lumazine protein (LumP) is a fluorescent accessory protein having 6,7-dimethyl-8-(1′-d-ribityl) lumazine (DMRL) as its authentic chromophore. It modulates the emission of bacterial luciferase to shorter wavelengths with increasing luminous strength. To obtain structural information on the native structure as well as the interaction with bacterial luciferase, we have determined the crystal structures of LumP from Photobacterium kishitanii in complexes with DMRL and its analogues, riboflavin (RBF) and flavin mononucleotide (FMN), at resolutions of 2.00, 1.42, and 2.00 Å. LumP consists of two β barrels that have nearly identical folds, the N-terminal and C-terminal barrels. The structures of LumP in complex with all of the chromophores studied are all essentially identical, except around the chromophores. In all of the structures, the chromophore is tethered to the narrow cavity via many hydrogen bonds in the N-terminal domain. These are absent in the C-terminal domain. Hydrogen bonding in LumP-FMN is decreased in comparison with that in LumP-RBF because the phosphate moiety of FMN protrudes out of the narrow cavity. In LumP-DMRL, the side chain of Gln65 is close to the ring system, and a new water molecule that stabilizes the ligand is observed near Ser48. Therefore, DMRL packs more tightly in the ligand-binding site than RBF or FMN. A docking simulation of bacterial luciferase and LumP suggests that the chromophore is located close enough for direct energy transfer to occur. Moreover, the surface potentials around the ligand-binding sites of LumP and bacterial luciferase exhibit complementary charge distributions, which would have a significant effect on the interaction between LumP and luciferase.Bioluminescent organisms are widely distributed in nature and comprise a remarkably diverse set of species (8, 11, 40). Among them, luminous bacteria have long been known to exist (15). Many studies of bacterial luminescence have been reported (34-36), and its biochemical mechanism has been the subject of many investigations (13, 14). The luminous bacteria illuminate using bacterial luciferases, which emit blue-green light through catalytic oxidation of reduced flavin mononucleotides (FMN) with long-chain aliphatic aldehydes. The maximal emission wavelengths (475 to 486 nm) of some Photobacterium strains are blue shifted with respect to those of purified luciferase (495 nm). This blue shift is induced by a fluorescent accessory protein called lumazine protein (LumP; 21 kDa) (22, 28, 29). LumP was first isolated from the luminescent marine bacterium Photobacterium phosphoreum (38) and later from Photobacterium leiognathi (28). Interestingly, LumP not only decreases the emission wavelength but also enhances the intensity of the light (29, 32). LumP possesses a noncovalently bound fluorophore, 6,7-dimethyl-8-ribityllumazine (DMRL), which is known to be the direct biosynthetic precursor of riboflavin (RBF) (10). Another accessory protein of bacterial luciferase, the yellow fluorescent protein (YFP; 23 kDa), was found in Vibrio fischeri Y-1. YFP contains RBF or FMN as a chromophore and modulates the emission wavelength of bacterial luciferase to yellow light (∼540 nm) (3, 7, 25, 33). LumP and YFP share amino acid sequence homology (37%). These fluorescent accessory proteins are believed to interact with bacterial luciferases in the intermediate states during the luciferase reaction (29, 32) and to transform the chemical energy from the luciferase reaction into the excitation of their bound, fluorescent ligands. However, the detailed mechanisms of the interaction and the interactive process have not yet been understood.LumP shares a high amino acid sequence homology with RBF synthase, which generates one RBF molecule from two DMRL molecules (10, 12). RBF synthase has a homo-trimer. RBF synthase consists of two segments with similar sequences and two closely related folds, the N-terminal and C-terminal domains (12, 24, 26, 39). In the crystal structure, each domain contained one DMRL derivative molecule. It is proposed that the catalytic site is located at the interface between the N-terminal domain of one subunit and the C-terminal domain of the adjacent subunit. Recently, Chatwell et al. reported the crystal structure of the lumazine protein of P. leiognathi (LumP_PL) in complex with RBF at a resolution of 2.5 Å (5). The overall structure is similar to that of RBF synthase. RBF fits into a narrow cavity in the N-terminal domain, but no ligand was observed in the C-terminal domain. It should be noted that they determined the crystal structure of a mutant protein (L49N) whose ability to bind to DMRL and RBF is partially impaired (16). In addition, it is known that LumP modulates the color of light generated by bacterial luciferase only when it is complexed with DMRL. Therefore, structural information on the wild-type LumP-DMRL complex is required to reveal the details of the ligand-binding mode of LumP at the LumP-luciferase interaction site.Here, we present the crystal structures of LumP from P. kishitanii (LumP_PK) in complexes with the authentic chromophore, DMRL, and its analogues, RBF and FMN, at 2.00-, 1.42-, and 2.00-Å resolutions. These are the highest-resolution structures that have been determined for this family of proteins. In addition, a docking simulation of bacterial luciferase and LumP_PK suggested that their chromophores are located closely enough for direct energy transfer to occur.