title: 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.
authors.affiliation: Graduate Program of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar), Rodovia João Leme dos Santos, km 110, Itinga, Sorocaba, SP, Brazil. viviani@ufscar.br
authors.forename: V R
authors.initials: VR
authors.lastname: Viviani
authors.forename: R A
authors.initials: RA
authors.lastname: Prado
authors.forename: D R
authors.initials: DR
authors.lastname: Neves
authors.forename: D
authors.initials: D
authors.lastname: Kato
authors.forename: J A
authors.initials: JA
authors.lastname: Barbosa
mesh_terms: D000214:Acyl Coenzyme A / Q000096:biosynthesis; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics; D019143:Evolution, Molecular; D049454:Firefly Luciferin / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates / Q000737:chemistry; D011485:Protein Binding; D013712:Tenebrio / Q000201:enzymology
chemical_list: D000214:Acyl Coenzyme A; D005456:Fluorescent Dyes; D019476:Insect Proteins; D009282:Naphthalenesulfonates; C501929:luciferyl-coenzyme A; D049454:Firefly Luciferin; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D008156:Luciferases; D003066:Coenzyme A Ligases