Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain.
Lumazine protein is believed to serve as an optical transponder in bioluminescence emission by certain marine bacteria. Sequence arguments suggest that the protein comprises two similarly folded riboflavin synthase-type domains, but earlier work also suggested that only one domain binds 6,7-dimethyl-8-ribityllumazine (DMRL). We show that the replacement of serine-48 or threonine-50 in the N-terminal domain of lumazine protein of Photobacterium leiognathi modulates the absorbance and fluorescence properties of bound DMRL or riboflavin. Moreover, the replacement of these amino acids is accompanied by reduced ligand affinity. Replacement of serine-48 by tryptophan shifts the (13)C NMR signal of the 6-methyl group in bound DMRL upfield by 2.9 ppm as compared to the wild-type protein complex. Replacement of threonine-50 causes a downfield shift of approximately 20 ppm for the (15)N NMR signal of N-5, as well as an upfield shift of 3 ppm for the (13)C NMR signal of C-7 in bound DMRL, respectively. The replacement of the topologically equivalent serine-144 and proline-146 in the C-terminal domain had no significant impact on optical properties, chemical shifts and apparent binding constants of bound DMRL. These data show that the N-terminal domain is the unique site for ligand binding in lumazine protein.
Illarionov
Boris
B
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany.
Eisenreich
Wolfgang
W
Wirth
Martina
M
Yong Lee
Chan
C
Eun Woo
Young
Y
Bacher
Adelbert
A
Fischer
Markus
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005454:Fluorescence Polarization; D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010776:Photobacterium / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C026648:lumazine protein, Photobacterium