Thr226 is a key residue for bioluminescence spectra determination in beetle luciferases

The comparison of click beetle and railroadworm luciferases (pH-insensitive ) with firefly luciferases (pH-sensitive) showed a set of conserved residue s differing between the two groups which could be involved with the biolumi nescence spectra pH sensitivity. The substitution C258V in Pyrocoelia miyak o (Pml) firefly luciferase and V255C in Ragophthalmus ohbai railroad worm l uciferase (Rol) had no effect on the bioluminescence spectra. Substitution of Thr226 in the green-light-emitting luciferases of Rol and Pyrearinus ter mitilluminans (Pyt) click beetle luciferases resulted in red-shifts (12 to 35 nm), whereas the substitution T226N in the red-light-emitting luciferase of Phrixothrix hirtus (PhRE) railroadworm resulted in a 10 nm blue-shift. In PmL the substitution N230S resulted in a typical red mutant (lambda (max ) = 611 nm). The bioluminescence spectrum of all these luciferase mutants d id not show altered pH-sensitivity nor considerably changed half-bandwidth in relation to the wildtype luciferases. Altogether present data suggest th at Thr226 is an important residue for keeping active-site core in both grou ps of beetle luciferases. The mechanism for bioluminescence color determina tion between pH-sensitive and pH-insensitive luciferases could be different . (C) 2001 Academic Press.