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

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of late ral lanterns along the body and red light through two cephalic lanterns. Th e cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda(max) = 542 nm), and for the head lanterns of P. hi rtus, which emit the most red-shifted bioluminescence (lambda(max) = 628 nm ) among luminescent beetles, were cloned. Positive clones which emitted gre en (Pv(GR): lambda(max) = 549 nm) and red(Ph-RE: lambda(max) = 622 nm) biol uminescence 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 l uciferases, respectively, and 47-49% with click-beetle luciferases. Ph-RE l uciferase has some unique residues which replace invariant residues in othe r beetle luciferases. The additional residue Arg 352 in Ph-RE, which is del eted in Pv(GR) polypeptide, seems to be another important structural featur e associated with red light production. As in the case of other railroad-wo rms and click-beetle luciferases studied, Phrixothrix luciferases do not un dergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shar ed in common between railroad-worm and click-beetle luciferase.