THE STRUCTURAL DETERMINANTS OF BIOLUMINESCENCE COLORS IN RAILROAD WORM AND OTHER pH-INSENSITIVE LUCIFERASES

摘要

Beetle luciferases are divided in two large groups according to their bioluminescence spectral sensitivity to pH: (Ⅰ) the pH-sensitive luciferases (fireflies) which suffer red-shift upon decreasing pH and other denaturing conditions and (Ⅱ) pH-insensitive (click beetle and railroad worms) luciferases which are not affected by these conditions. Most studies on the relationship between structure and emission spectra have focused on the pH-sensitive luciferases, much less is known about pH-insensitive luciferases. We cloned the cDNAs for four pH-insensitive luciferases: the red and green light-emitting luciferases of the Brazilian Phrixothrix hirtus (PxhRE) and P. vivianii railroad worms (PxvGR), the green emitting luciferases of the Brazilian Pyrearinus termitilluminans larval click beetle (Pte) and the Japanese Ragophthalmus ohbai star worm (Rol). We found that the region before residue 344 determines the bioluminescence spectrum in railroad worm luciferases and that the residues R215 and T226 in pH-insensitive luciferases (N229 in pH-sensitive luciferases) are key residues for green light emission. Through the construction of new chimerae using Phrixothrix luciferases we here show that the region between residues 220-344 is the main determinant of bioluminescence colors in railroad worm luciferases. We also investigated the effect of single and double substitutions of some conserved residues differing between pH-sensitive and pH-insenitive luciferases.