Enzymatic promiscuity and the evolution of bioluminescence

Abstract

Bioluminescence occurs when an enzyme, known as a luciferase, oxidizes a small-molecule substrate, known as a luciferin. Nature has evolved multiple distinct luciferases and luciferins independently, all of which accomplish the impressive feat of light emission. One of the best-known examples of bioluminescence is exhibited by fireflies, a class of beetles that use d-luciferin as their substrate. The evolution of bioluminescence in beetles is thought to have emerged from ancestral fatty acyl-CoA synthetase (ACS) enzymes present in all insects. This theory is supported by multiple lines of evidence: Beetle luciferases share high sequence identity with these enzymes, often retain ACS activity, and some ACS enzymes from nonluminous insects can catalyze bioluminescence from synthetic d-luciferin analogues. Recent sequencing of firefly genomes and transcriptomes further illuminates how the duplication of ACS enzymes and subsequent diversification drove the evolution of bioluminescence. These genetic analyses have also uncovered candidate enzymes that may participate in luciferin metabolism. With the publication of the genomes and transcriptomes of fireflies and related insects, we are now better positioned to dissect and learn from the evolution of bioluminescence in beetles.

Keywords: adenylation; beetles; bioluminescence; chemistry; enzymes; evolution; firefly; luciferase; luciferin; metabolism.

Figure 1.

Firefly luciferase (Fluc) retains ancestral ACSL activity. a) Fluc catalyzes the adenylation and oxidation of D-luciferin to release a photon of light; b) Fluc can also ligate coenzyme A to fatty acids. Both reactions involve a key adenylated intermediate. Adapted from Mofford et al. [13].

Figure 2.

Mechanism of beetle bioluminescence. a) D-luciferin is adenylated, which makes the C4 carbon more acidic. The carbanion reduces molecular oxygen, forming a C4 radical and superoxide. Recombination of the radicals forms a peroxide that then attacks the AMP ester to form a strained dioxetanone ring. The oxygen-oxygen bond breaks, releasing carbon dioxide and forming an excited-state oxyluciferin, which radiatively relaxes to the ground state; b) In a “dark” reaction pathway, the C4 radical intermediate can instead form dehydroluciferyl-AMP, which is incapable of further oxidation and light emission.

Figure 3.

Alignment of firefly luciferase with homologous insect fatty acyl-CoA synthetases. Photinus pyralis firefly luciferase (Fluc), Agrypnus binodulus luciferase-like protein (AbLL), Drosophila melanogaster long-chain fatty acyl-CoA synthetase CG6178, and Pyrophorus angustus luciferase-like protein (PaLL) aligned using MUSCLE [30]. Coloring indicates similarity in sequence, with darker shades of blue representing more similarity. Black asterisks under the lysine residues 443 and 529 (Fluc numbering) indicate catalytically important residues detailed in the text. The red vertical line and corresponding asterisk indicates where the N-terminal domains and C-terminal domains meet in the enzymes. Below the alignment are luciferin structures and the enzymes that can catalyze light emission from them.

Figure 4.

The luciferase from the firefly Photinus pyralis (left) can accept both D-luciferin and synthetic luciferins as luminogenic substrates, and retains ancestral long-chain fatty acyl-CoA synthetase activity. The homologous long-chain fatty acyl-CoA synthetase AbLL from the non-luminescent beetle Agrypnus binodulus (right) is unable to emit light when treated with D-luciferin.

Figure 5.

Luciferin metabolism. a) Arbutin is hydrolyzed to release 1,4 hydroquinone, which is then oxidized to p-benzoquinone. Subsequent reaction with L-cysteine yields 2-S-cysteinylhydroquinone. In a poorly characterized series of steps, formal decarboxylation and the addition of second molecule of L-cysteine yields L-luciferin; b) Formation of D-luciferin is thought to proceed using the fatty acyl-CoA synthetase activity of firefly luciferase to enantioselectively thioesterify L-luciferin to L-luciferyl CoA, followed by epimerization and subsequent thioester hydrolysis to yield D-luciferin; c) A putative luciferin regenerating enzyme (LRE) might convert oxyluciferin back to L-luciferin via a 2-cyano-6-hydroxybenzothiazole intermediate; d) The 6’-hydroxyl of both D- and L-luciferin can be reversibly sulfated by luciferin sulfotransferase (LST). It is unknown whether LST can utilize oxyluciferin as a substrate.

Figure 6.

Adenylating enzymes can operate on diverse substrates, enhancing the potential for identifying new examples of luciferase activity. a) The plant Arabidopsis thaliana encodes enzymes related to fatty acyl-CoA synthetases that operate on bulky intermediates in the jasmonate biosynthetic pathway. For example, the enzyme At5g63380 can catalyze the conversion of 12-oxo-phytodienoic acid (OPDA) to OPDA-CoA; b) Fridericia earthworms possess an as-yet unidentified ATP-dependent luciferase activity based on a structurally distinct luciferin. The site of oxidation is shown in red.

Figure 7.

Examples of luciferins that do not require adenylation for activation. a) Coelenterazine and vargulin share a common luminescent imidazopyrazinone scaffold, shown in blue. Other distinct luminescent scaffolds include dinoflagellate and fungal luciferins; b) The mechanism for bioluminescence from coelenterazine and marine luciferases. Molecular oxygen is shown in red to illustrate the fate of each oxygen atom in the ensuing chemistry.