Interconversion of ketoprofen recognition in firefly luciferase-catalyzed enantioselective thioesterification reaction using from Pylocoeria miyako (PmL) and Hotaria parvura (HpL) just by mutating two amino acid residues.
J Biotechnol
; 168(3): 277-83, 2013 Nov.
Article
em En
| MEDLINE
| ID: mdl-23685028
ABSTRACT
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.
Palavras-chave
2-Arylpropanoic acid; CoASH; Enantioselective thioesterification; Firefly luciferase; Hotaria parvura; HpL; LUC-H; LcL; LmL; MD; Molecular dynamics calculation; PPB; PmL; PpL; Pylocoeria miyako; Substrate recognition; coenzyme A; firefly luciferase from Hotaria parvura; firefly luciferase from Luciola cruciate; firefly luciferase from Luciola lateralis; firefly luciferase from Luciola mingrelica; firefly luciferase from Photinus pyralis; firefly luciferase from Pylocoeria miyako; molecular dynamics; potassium phosphate buffer
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cetoprofeno
/
Vaga-Lumes
/
Luciferases de Vaga-Lume
Limite:
Animals
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article