Let there be light

Brazilian researchers discover one the “switches” that can make some enzymes used in the biomedical and biotech fields become luminescent.

By Elton Alisson

Agência FAPESP – The researchers of the Bioluminescence and Biophotonic Group at Universidade Federal de São Carlos (UFSCar- Sorocaba campus), took an important step toward making some enzymes utilized by the biomedical and biotechnological field emit light.

The property is important for studying diseases like cancer or bacterial infections, for example. Scientists discovered that one of the main “breakers” in the “light box” of enzymes with low luminescent capacity of the same class of luciferase – responsible for emitting cold light and visible in fireflies – could be modified to increase the intensity of their light.

The discovery, the result of a research project on Luciferases of Coleoptera: Structural and Functional Evolution and Engineering for Biotechnological Uses funded by FAPESP under the Regular Research Awards – will be published by the end of this month in Photochemical and Photobiological Sciences magazine.

In 2009, the same group cloned and isolated an enzyme of the same family of luciferase (AMP-CoA-ligases) from the larva of a non-luminescent insect (beetle). The AMP-CoA-ligases enzyme, weakly luminescent and known as a protoluciferase, was used to study how firefly luciferase develop the capacity to catalyze the oxidation reaction of luciferin – the compound responsible for bioluminescence in insects – and produce intense visible light.

In the last few years, in comparing the sequences of amino acids of protoluciferase with luciferase, the UFSCar researchers began to identify parts of the structures of both that could be involved in determining the activity of producing light.

Through genetic engineering techniques, the doctoral student Rogilene Prado and researcher Vadim Viviani, made mutations to the amino acids of protoluciferase. Now, the group identified that the mutation of one of these amino acids significantly increases the luminescent activity of the enzyme, making it very similar to that of luciferase.    

“It is as if the protoluciferase enzyme were an electronic circuit that has a battery represented by oxygen and a light bulb, which is the lucerfin. This time, we discovered one of the main switches present in the structure of enzymes, which is responsible for turning on the light bulb’s batteries. Or rather, it prompts the reaction between luciferin and oxygen, adding light,” Viviani explains to Agência FAPESP.  
 
According to him, the discovery makes it possible to turn other enzymes in the AMP-CoA-ligases family, which are used in biomedical, biotechnological and environmental areas that do not produce light in luminescents.

Found in all organisms including bacteria and humans, AMP-CoA-ligases perform varied metabolic functions, like biosynthesis of pigments (in plants), metabolism of lipids, synthesis of antibiotics and elimination of toxic substances and foreign chemical compounds to an organism or the biological system (xenobiotics).

The first reaction that all catalyze is activation of organic acids, like amino acids, fatty acids and the luciferin of fireflies, which is oxidized by luciferase, producing light. As a result, the researchers intend to utilize them as indicators of certain organic acids used in biomedicine, like toxic acids, and biotechnology.

“The capacity of serving as an indicator to select certain organ acids under in the pharmaceutical and biotechnology fields perhaps represents the greatest potential application of these enzymes,” says Viviani.

Evolution in the laboratory

According to the project coordinator, a few luciferases of North American, European and Japanese fireflies are used as analytic reactors. They are used to detect the metabolic state of a biological sample and biomarkers of genetic expression, or to mark cancer cells in biophotonic studies, for example.

Through research with the prototype of the luciferase enzyme that was cloned and increased in luminescence, the Brazilian researchers intend to create a new luciferase enzyme through genetic engineering that has the property of emitting light comparable to the luciferases currently utilized on the market.

“With the ideal conditions for evolution, this protoluciferase could become a luciferase. We are simulating its evolution in the laboratory,” says Viviani.

The USFCar research group is one of the only teams dedicated to the study of luciferase enzymes in Brazil. At the Chemistry Institute at Universidade de São Paulo (USP), there is another group, coordinated by Cassius Stevani, with whom USFCar collaborates for study of luminescent fungi.

Around the globe, the research groups in the area are largely established in the United States, Europe and Japan – the latter collaborates with Brazilian researchers. And, according to Viviani, none of them has yet managed to clone a protoluminiscent enzyme capable of emitting light similar to the Brazilian group.

An abstract of the study “Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site,” authored by Professor Viviani and other researchers can be read at: pubs.rsc.org/en/Content/ArticleLanding/2011/PP/c0pp00392a.