THE INFLUENCE OF THE LOOP BETWEEN RESIDUES 223-235 IN BEETLE LUCIFERASES BIOLUMINESCENCE SPECTRA
V. R. Viviani1,2; Frederico G.C. Arnoldi1; A.J. Silva Neto1; M. R. Brochetto-Braga1; and João A.R.G. Barbosa3
1Dept. Biologia Celular e Molecular, Instituto de Biociências, Universidade Estadual de São Paulo (UNESP), Rio Claro, SP, Brazil; 2Universidade Federal de São Carlos, Sorocaba, SP, Brazil and 3Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brazil
Beetle luciferases produce bioluminescence in the green-red range of the spectrum. They are divided in two functional groups, according to the bioluminescence spectral sensitivity to pH: the pH-sensitive firefly luciferases (Lampyridae) and the pH-insensitive click beetle (Elateridae) and railroadworm (Phengodidae) luciferases. Most studies focused on the pH-sensitive firefly luciferases. Despite the identification of important residues for bioluminescence color, the structural determinants of bioluminescence colors and pH-sensitivity remain enigmatic. Through a comparative site-directed mutagenesis study, using our cloned pH-sensitive (Cratomorphus, Macrolampis) and pH-insensitive luciferases (Phrixotrix spp, Pyrearinus termitilluminans), we found that several key residues for bioluminescence color and pH-sensitivity determination are grouped in the loop between residues 223-235. Besides the residue N229 in the pH-sensitive luciferases and the respective T226 in the pH-insensitive luciferases, the position 227 was found to be involved in labile interactions, its substitution resulting in time-dependent spectral shifts. The results are consistent with the structural key role of the loop between residues 223-235 in bioluminescence color determination in beetle luciferases. (Financial support: FAPESP)
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