Mutagenesis of firefly luciferase shows that cysteine residues are not required for bioluminescence activity

Abstract

Conflicting reports exist in the literature as to whether sulfhydryl groups are essential for firefly luciferase activity. Inactivation of Photinus pyralis luciferase with ./V-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and site-directed mutagenesis demonstrate that the cysteine residues are not absolutely required for activity. However, loss of P. pyralis luciferase activity is observed when any of the 4 cysteine residues is replaced with serine.© 1997 Federation of European Biochemical Societies.Key words: Photinus pyralis; Oxygenase; Active site; Protein modification; Amino acid sequence homology of the essentiality of sulfhydryl groups in firefly luciferase has been reexamined using a combination of iV-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and site-specific mutagenesis to modify the cysteine residues in Pp luciferase [10] and in the luciferases of the Japanese fireflies, Hotaria parvula (Hp) [11] and Pyrocoelia miyako (Pm) [11]. The results show that, even though modification of the cysteine residues can cause significant changes in activity, sulfhydryl groups are not absolutely required for activity.

Cloning and Expression of the Bioluminescent Photoprotein Pholasin from the Bivalve Mollusc Pholas dactylus

“…In addition, there was close sequence homology between the cysteine domains of the luciferases in seven species of firefly. However, firefly luciferase is active after cysteine modification (47). It is also interesting to note that pholasin contains no histidine residues.…”

The origin, diversity, and structure function relationships of insect luciferases

“…Two sulfhydryl groups in firefly luciferase were originally suggested to be important for catalysis in bioluminescence [113,114], however, recent investigations show that no sulfhydryl group is essential for activity [115,116]. The deletion of the last C-terminal 12 residues in firefly luciferase results in complete loss of activity [117], showing that the C-terminal domain is essential for activity.…”

Discrimination Between Defects in Elongation Fidelity and Termination Efficiency Provides Mechanistic Insights into Translational Readthrough

“…We chose to utilize firefly luciferase to develop this misincorporation reporter system due to the availability of its crystal structure and the many genetic studies previously carried out with this protein. [14][15][16][17][18][19][20][21] In those studies, it was shown that the His residue encoded at position 245 (His245 (CAC)) and the Lys residue encoded at position 529 (Lys529 (AAA)) each play an important role in the enzymatic activity of firefly luciferase, [15][16][17] probably because these residues properly orient the substrate and stabilize the transition state of the enzyme. It was previously reported that Lys529 is essential for enzymatic activity, 17 while His245 is important, but not absolutely essential.…”