The Role of Lysine 529, a Conserved Residue of the Acyl-Adenylate-Forming Enzyme Superfamily, in Firefly Luciferase

Abstract

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen in a two-step process. The enzyme first catalyzes the adenylation of the carboxylate substrate luciferin with Mg-ATP followed by the oxidation of the acyl-adenylate to the light-emitting oxyluciferin product. The beetle luciferases are members of a large family of nonbioluminescent proteins that catalyze reactions of ATP with carboxylate substrates to form acyl-adenylates. Formation of the luciferase-luciferyl-AMP complex is a specific example of the chemistry common to this enzyme family. Site-directed mutants at positions Lys529, Thr343, and His245 were studied to determine the effects of the amino acid changes at these positions on the individual luciferase-catalyzed adenylation and oxidation reactions. The results suggest that Lys529 is a critical residue for effective substrate orientation and that it provides favorable polar interactions important for transition state stabilization leading to efficient adenylate production. These findings as well as those with the Thr343 and His245 mutants are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

Thermostabilization of Firefly Luciferases Using Genetic Engineering

“…These models were further confirmed by the independent studies: the residual activity of the luciferase mutant, in which Lys529 was changed to Ala, was less than 0.1% (Branchini et al, 2000). The role of others residues of the luciferase active site was supported by mutagenesis methods (Branchini et al, 1999(Branchini et al, , 2000(Branchini et al, , 2001(Branchini et al, , 2003. Fig.…”

A comprehensive analysis of translational missense errors in the yeastSaccharomyces cerevisiae

“…Fluc is the misreading reporter; Rluc serves as an internal control for protein abundance. Lysine-529 (K529) of Fluc is highly conserved among firefly luciferases (Ye et al 1997) and even among the more highly diverged superfamily of adenylate-forming enzymes (Branchini et al 2000). Point mutations in K529 reduce enzymatic activity up to 1600-fold in bacteria (Branchini et al 2000;Kramer and Farabaugh 2007).…”

“…To better understand the true in vivo range of misreading errors, we developed an assay system that measured the frequency of every near-cognate misreading error by E. coli tRNA Lys UUU (Kramer and Farabaugh 2007). The system uses mutant forms of a firefly luciferase (Fluc) reporter gene with inactivating mutations in the essential active-site lysine-529 (Branchini et al 2000). The proteins expressed from these mutant genes have low but measurable activity, which could be explained in two ways.…”

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

“…Indeed, studies with wild-type and red mutant luciferase from Luciola cruciata showed that native luciferase adopts a ''closed form'' during the formation of the high-energy intermediate responsible for light emission, creating a hydrophobic pocket around the active site, and an ''open form'' in complex with the reactants and products (6). The major part of the amino acid residues important to the bioluminescent activity is located in the N-terminal portion and only one in the C-terminal domain (7), and it was demonstrated that luciferase can produce light bearing only the N-domain, albeit with a luminescent output of only 0.03% of the complete enzyme (8).…”