Complementation of subunits from different bacterial luciferases. Evidence for the role of the beta subunit in the bioluminescent mechanism.
Complementation of the nonidentical subunits (alpha and beta) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (alpha h beta p) containing the alpha subunit from B. harveyi luciferase (alpha h) and the beta subunit from P. phosphoreum luciferase (beta p). The complementation was unidirectional; activity could not be restored by complementing the alpha subunit of P. phosphoreum luciferase with the beta subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the alpha subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH2 and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the Kd for FMNH2 actually being closer to that observed for the P. phosphoreum luciferase (the source of the beta subunit). These results provide direct evidence that modification or alteration of the beta subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the beta subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the alpha and beta subunits are involved with the initial interaction with FMNH2, whereas subsequent steps in the mechanism are dictated exclusively by the alpha subunit and are unaffected by alterations in the beta subunit.
D005486:Flavin Mononucleotide / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism*; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010776:Photobacterium / Q000201:enzymology*; D055503:Protein Multimerization; D013045:Species Specificity; D014733:Vibrio / Q000201:enzymology*; D014737:Vibrionaceae / Q000201:enzymology*
D046911:Macromolecular Substances; D005486:Flavin Mononucleotide; D008156:Luciferases