A Route from Darkness to Light: Emergence and Evolution of Luciferase Activity in AMP-CoA-Ligases Inferred from a Mealworm Luciferase-like Enzyme

Abstract

The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of I327T/S mutations and N-terminal fusion, the luminescence activity of this enzyme was increased to visible levels, with the development of a totally new orange-emitting luciferase. These results open the possibility of engineering luciferase activity in a set of AMP-CoA-ligases.

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

“…However, all insects express longchain fatty acyl-CoA synthetases (ACSLs) that share high homology to beetle luciferases and are hypothesized to be their evolutionary antecedents (4)(5)(6). These two classes of enzymes are both members of the adenylate-forming superfamily (7) and share the ability to make AMP esters of fatty acids as well as the ability to displace the AMP ester with CoASH ( Fig.…”

“…Beetle luciferases differ from other insect ACSLs in their ability to chemically generate light by adenylating and oxidizing D-luciferin, a small molecule naturally found in bioluminescent beetles. How this additional activity developed is unknown, although weak bioluminescence has been reported by treating a beetle ACSL with D-luciferin (6,9).…”

Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado

“…The cDNA of the luciferase-like ligase was cloned, and the enzyme structure and function determined by Viviani et al (2013). The authors concluded that luciferases may have evolved from ligases by mutagenesis of amino acids located in the activity site that enable them to act as a luminogenic monoxidase.…”

“…The authors concluded that luciferases may have evolved from ligases by mutagenesis of amino acids located in the activity site that enable them to act as a luminogenic monoxidase. Mutations of the so-called "protoluciferases" may engender new chemiluminescence-eliciting ligases, capable of using various carboxylic acids as substrates and drawing forth different emission colors (Prado et al 2011, Viviani et al 2013. These findings may significantly expand the analytical applications of engineered luciferases and AMP-CoA ligases (Roda 2011, Li et al 2010, Shimomura 2012.…”

Living Light in the Darkness: Facts and Stories

“…[44][45][46] Em ambos os casos -luciferase e ligase -, o papel do ATP é ativar a carboxila na forma de um adenilado (anidrido misto), um excelente grupo de partida em ataques nucleofílicos: à carbonila no caso da luciferase, ou um tioéster nos casos de proteínas e gliceróis, também um excelente g upoà deà saídaà aoà ata ueà doà α-aminogrupo do aminoácido a ser inserido na cadeia crescente do peptídeo, ou do grupo hidroxila do glicerol receptor do grupo acila. 45 A propósito, em anos recentes foi relatada a ação catalítica de luciferase sobre a acilação de glicerol por ácidos graxos, permitindo o desenvolvimento de métodos analíticos para a determinação de ácidos carboxílicos e triacilgliceróis em soro.…”