The N-terminal amino acid sequences of the firefly luciferase are important for the stability of the enzyme.
The structural and catalytic role of the N-terminal amino acid sequence of Photinus pyralis luciferase was investigated by site-directed mutagenesis. The firefly luciferase activity of a series of deletion and site-directed mutants in the amino-terminal region was investigated in vitro and in vivo. The mutant luciferases were produced either by in vitro transcription and translation or by expressing the cDNA encoding firefly luciferase in the yeast Saccharomyces cerevisiae as a fusion protein to the galactose DNA binding domain protein. Deleting the N-terminal amino acid residues from 3 to 10 dramatically reduced the luciferase activity to less than 1% of the wild-type activity. A marked decrease in the activity (to < 5%) was also observed with the Lys8Glu mutant. The Gly10Arg and Pro11Ala mutants showed 20-30% activity compared to the wild type. On the other hand, mutant Asn6Lys retained the wild-type activity level. Randomizing 3-11 N-terminal amino acids also showed a marked decrease in activity (< 1%). The mutant luciferases with extremely low levels of enzymatic activity were thermally unstable. These mutational and stability data correlate with the crystal structure of firefly luciferase in which specific amino acids in the N-terminal region form hydrogen bonds to the amino acids in the neighboring beta-stranded sheet. Because the N-terminal region is not part of the active site, the present results suggest that the highly conserved N-terminal amino acid sequences of the firefly luciferase are important for stabilizing the protein in its proper conformation for optimal enzyme activity.
Sung
D
D
Kumho Life & Environmental Science Laboratory, Kwangju, Korea.
Kang
H
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001517:Coleoptera / Q000201:enzymology*; D004795:Enzyme Stability; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D008156:Luciferases