Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.
Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.
Branchini
B R
BR
Department of Chemistry, Connecticut College, New London 06320, USA. brbra@conncoll.edu
Magyar
R A
RA
Murtiashaw
M H
MH
Anderson
S M
SM
Helgerson
L C
LC
Zimmer
M
M
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D001517:Coleoptera / Q000201:enzymology*; D006639:Histidine / Q000235:genetics; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008239:Lysine / Q000235:genetics; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000235:genetics
D019476:Insect Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D013912:Threonine; D006639:Histidine; D008156:Luciferases; D008239:Lysine