Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases:: Relationship between bioluminescence spectra and primary structures

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda(max) = 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda(max) = 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (Pv(GR): lambda(max) = 549 nm) and red(Ph-RE: lambda(max) = 622 nm) bioluminescence were isolated. The lucifereases coded by Pv(GR) (545 amino acid residues) and Ph-RE (546 amino acid residues) cDNAs share 71% identity. Pv(GR) and Ph-RE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. Ph-RE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in Ph-RE, which is deleted in Pv(GR) polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.