RESUMO
A simple reaction procedure for chemiluminescence of firefly luciferin (D-luc) using n-propylphosphonic anhydride (T3P) is reported. A luminescent photon is produced as a result of one-pot reaction, only requiring mixing with the substrate carboxylic acid and T3P in the presence of a mild organic base.
Assuntos
Luciferina de Vaga-Lumes/química , Luminescência , Organofosfonatos/química , Propano/análogos & derivados , Alcinos/química , Animais , Antracenos/química , Biomimética , Ácidos Carboxílicos/química , Cromatografia Líquida de Alta Pressão , Etilaminas/química , Vaga-Lumes , Luciferina de Vaga-Lumes/análogos & derivados , Ácidos Indolacéticos/química , Estrutura Molecular , Processos Fotoquímicos , Fótons , Propano/química , Análise Espectral , Ureia/análogos & derivados , Ureia/químicaRESUMO
We identified the critical amino acid residues for substrate recognition using two firefly luciferases from Pylocoeria miyako (PmL) and Hotaria parvura (HpL), as these two luciferase enzymes exhibit different activities toward ketoprofen. Specifically, PmL can catalyze the apparent enantioselective thioesterification reaction, while HpL cannot. By comparing the amino acid sequences around the active site, we identified two residues (I350 and M397 in PmL and F351 and S398 in HpL) that were different between the two enzymes, and the replacement of these amino acids resulted in changing the ketoprofen recognition pattern. The inactive HpL was converted to the active enzyme toward ketoprofen and vice versa for PmL. These residues also affected the enantioselectivity toward ketoprofen; however, the bioluminescent color was not affected. In addition, using molecular dynamics calculations, the replacement of these two amino acids induced changes in the state of hydrogen bonding between ketoprofen and the S349 side chain through the active site water. As S349 is not considered to influence color tuning, these changes specifically caused the differences in ketoprofen recognition in the enzyme.