Formation of hybrid luciferases from subunits of different species of Photobacterium.

Enzyme divergence within three species of the genus Photobacterium (P. fischeri, P. leiognathi, and P. phosphoreum) was studied by comparing the catalytic characteristics and quaternary interactions of bacterial luciferases isolated from each species. Each luciferase was composed of two subunits of different molecular weights as determined by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Subunits were isolated in quantity by DEAE-Sephadex gel filtration in 7 M urea. Isolated subunits had no luciferase activity after renaturation in buffer, but active enzyme could be recovered by renaturation of the heavy and light subunits together. Renaturation of hybrid pairs (containing one subunit from each of two different species) yielded active luciferases, but only in cases where a heavy subunit of one species was combined with a light subunit of another. These hybrids exhibited in vitro catalytic characteristics most like those of the parent luciferase from which the heavy subunit was derived. The light subunit of P. leiognathi luciferase conferred an increased thermal stability to all enzymes containing it. The heavy subunit of each of the three Photobacterium species was sensitive to trypsinization. Thus, on the basis of structural and functional analogies with the luciferase from Beneckea harveyi, the heavy and light subunits of Photobacterium species have been designated alpha and beta, respectively.