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Biological Chemistry

Deciphering the color code of bioluminescence

Two amino acid residues help tune the wavelength of light produced by luciferase

by Erika Gebel Berg
August 23, 2016

Credit: Robert Sisson/National Geographic Creative
A railroad worm glows bright thanks to the bioluminescent protein luciferase. Subtle structural differences in the protein’s active site affect the color.

The luminescent protein luciferase, found in fireflies and other beetles, is a bioanalytical workhorse. Scientists use it as a reporter molecule to detect gene expression, microbial contamination, and other biological processes.

Luciferase comes in different colors depending on the insect species of origin. However, scientists have been at a loss for how luciferase proteins produce their color variety. Now, researchers have identified two amino acids in luciferase that determine whether an insect glows green or red (Biochemistry 2016, DOI:10.1021/acs.biochem.6b00260). This insight may someday help scientists to engineer enzymes that glow in colors beyond the palette produced by wild creatures.

Luciferase generates light by catalyzing the oxidation of the compound luciferin, creating an unstable product, oxyluciferin, which decays to release a photon of light. The energy of that light determines the hue, but what determines the energy? To answer that question, Vadim R. Viviani of Federal University of São Carlos and colleagues compared the luciferase from fireflies, which makes greenish light, to a version in a type of beetle larvae known as railroad worms that produces red light. Previous studies on firefly luciferase pointed to the potential importance of two particular amino acids in tuning the wavelength of light emission, prompting Viviani to attempt to tease out the residues’ roles.

A previously published high-resolution crystal structure of firefly luciferase revealed that an arginine in position 337 and a glutamic acid in position 311 form a salt bridge near the enzyme’s active site. Viviani’s team made a series of mutations that disrupted this salt bridge and found that all the mutated enzymes produced redder light. This shows, Viviani says, that the salt bridge acts as a gate: Breaking the salt bridge allows water to flood the active site, altering the way the enzyme holds on to oxyluciferin and lowering the energy of emission, thus producing redder light. The salt-bridge hypothesis would also explain why firefly luciferase makes redder light in acidic conditions, since the salt bridge is broken down at low pH, Viviani says.

In contrast, railroad worm luciferase has a leucine in position 334 which corresponds to the arginine in firefly luciferase, but it doesn’t form a salt bridge with glutamic acid. Swapping the leucine for an arginine shifted the wavelength of the emitted light 11 nm towards the blue, supporting the importance of an arginine at this site for the emission of green light instead of red.

“The study complements previous work about the subtleties of the active site,” says Andrew M. Gulick of the University of Buffalo. “I think the salt bridge is influencing the light color, but I’m not sure it’s the key interaction. It certainly is important. I would like to see structural studies to look at these mutants and then be better able to see the atomic-scale results of these changes.”

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