A Tale Of Two Luciferins: Fungal and Earthworm New Bioluminescent

Sep 26, 2016 - (1) Bioluminescent systems of terrestrial luminous species such as insects,(3) worms,(4) ... 23, 24) in BRET pairs (bioluminescent reso...
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A Tale Of Two Luciferins: Fungal and Earthworm New Bioluminescent Systems Aleksandra S. Tsarkova,†,‡ Zinaida M. Kaskova,†,‡ and Ilia V. Yampolsky*,†,‡ †

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117997, Russia Pirogov Russian National Research Medical University, Ostrovitianova 1, Moscow 117997, Russia

‡

S Supporting Information *

CONSPECTUS: Bioluminescence, the ability of a living organism to produce light through a chemical reaction, is one of Nature’s most amazing phenomena widely spread among marine and terrestrial species. There are various different mechanisms underlying the emission of “cold light”, but all involve a small molecule, luciferin, that provides energy for light-generation upon oxidation, and a protein, luciferase, that catalyzes the reaction. Different species often use different proteins and substrates in the process, which suggests that the ability to produce light evolved independently several times throughout evolution. Currently, it is estimated that there are more than 30 different mechanisms of bioluminescence. Even though the chemical foundation underlying the bioluminescence phenomenon is by now generally understood, only a handful of luciferins have been isolated and characterized. Today, the known bioluminescence reactions are used as indispensable analytical tools in various fields of science and technology. A pressing need for new bioluminescent analytical techniques with a wider range of practical applications stimulates the search and chemical studies of new bioluminescent systems. In the past few years two such systems were unraveled: those of the earthworms Fridericia heliota and the higher fungi. The luciferins of these two systems do not share structural similarity with the previously known ones. This Account will survey structure elucidation of the novel luciferins and identification of their mechanisms of action. Fridericia luciferin is a key component of a novel ATPdependent bioluminescence system. Structural studies were performed on 0.005 mg of natural substance and revealed its unusual extensively modified peptidic nature. Elucidation of Fridericia oxyluciferin revealed that oxidative decarboxylation of a lysine fragment of luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as a light emitter. Along with luciferin, a number of its natural analogs were found in the extracts of worm biomass. They occurred to be highly unusual modified peptides comprising a set of amino acids, including threonine, aminobutyric acid, homoarginine, unsymmetrical N,N-dimethylarginine and extensively modified tyrosine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and raise novel questions concerning their biosynthetic origin. Also in this Account we discuss identification of the luciferin of higher fungi 3-hydroxyhispidin which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. Furthermore, it was shown that 3hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

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INTRODUCTION Over 40 years from the early 1950s until the late 1980s, a gradual and steady accumulation of knowledge of various biochemical systems responsible for generation of light in live organisms has considerably progressed.1 Detailed investigations of bioluminescent systems of various organisms have revealed that the mechanisms underlying visible light emission vary in many known bioluminescent species. That is to say that not only the substrates (termed luciferins) and various cofactors taking part in bioluminescent reactions are different, but the enzymes catalyzing these reactions vary as well. Out of approximately 30 known different bioluminescent mechanisms only a handful of © 2016 American Chemical Society

luciferins have been isolated and characterized (Figure 1), and for some of them the luciferase genes were also sequenced and cloned.2 Overall, all known bioluminescent mechanisms can be divided into two distinct classes depending on the enzyme involved: luciferase or photoprotein systems. Luciferin−Luciferase Systems

Reactions catalyzed by luciferases are classical reactions of separable enzyme and substrate, in which luciferin is oxidized by Received: June 28, 2016 Published: September 26, 2016 2372

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Figure 1. Structures of luciferins known before 2014.

Scheme 1. Luciferin−Luciferase Reaction Mechanism

and oxygenated coelenterazine (2-hydroperoxycoelenterazine). Decomposition of photoprotein into apoprotein, coelenteramide, and CO2 upon binding of calcium ions to photoprotein surface is accompanied by light emission (Scheme 2).12 As this reaction involves only one molecule, the amount of emitted light is directly proportional to the amount of photoprotein.

oxygen yielding electronically excited product - oxyluciferin, that relaxes to the ground state by photon emission (for structures of known oxyluciferins see Figure S1). In the case of luciferin− luciferase reaction, the intensity and duration of light emission depend on the amounts of both substrate and enzyme. The turnover of luciferase takes place repeatedly, completely utilizing luciferin.1 Bioluminescent systems of terrestrial luminous species such as insects,3 worms,4 and fungi,5 as well as of many marine organisms, such as various crustaceans,6,7 coelenterates,8 molluscs,9 bacteria,10 and fish belong to this class. A typical example of enzyme−substrate reaction is represented by the ostracod Cypridina system6 (Scheme 1):

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MODERN APPLICATIONS OF BIOLUMINESCENCE

The phenomenon of bioluminescence stays in the spotlight of researchers in a wide range of fields of science, including fundamental and applied biology, chemistry, and medicine. Due to the recent advances in light detection technologies,13 high quantum yields of bioluminescence and relative nontoxicity of luciferin reactions, a tremendous range of analytical techniques based on this phenomenon have been developed, most of which are now actively used in cancer studies,14 investigations of infectious diseases,15−18 and environmental monitoring.19,20 Luciferases are extensively used as convenient instruments in ATP tests,21 reporter gene analysis,22 biosensors,19,23,24 in BRET pairs (bioluminescent resonance energy transfer)25−27 and luciferase complementation assay28−30 for protein−protein and

Photoproteins, Ca2+-Triggered Mechanism

Photoprotein systems, common exclusively among marine organisms, are characterized by the absence of a separable substrate and a seeming lack of necessity for molecular oxygen during the bioluminescence reaction. These observations were first made on the jellyfish Aequorea.11 The following investigations have shown that most coelenterates utilize a common luciferin, coelenterazine. In photoprotein systems, a stable enzyme−substrate complex is formed between the apoprotein 2373

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Accounts of Chemical Research Scheme 2. General Mechanism of Bioluminescence Reaction of Photoproteins

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BIOLUMINESCENT SYSTEM OF EARTHWORM FRIDERICIA HELIOTA, HISTORY OF DISCOVERY For a long time, the chemistry of earthworm bioluminescence was considered to be uniform and utilyzed the same substrates. Indeed, until quite recently, all investigated oligochaetes displayed H2O2-dependent bioluminescence, giving crossreactions with each other, with the emission maxima ranging from green to yellow, depending on the species.52 Ten years ago two novel bioluminescent earthworm species belonging to the same family of Oligochaeta worms were discovered in Krasnoyarsk (Russia) by our collaborators: one of them was described as a new species Fridericia heliota, whereas the other was assigned to the Henlea genus.53 Despite their taxonomic proximity, these two species happened to have completely different chemical nature of bioluminescence, giving cross-reaction neither between each other nor with any other known bioluminescent system. The most distinctive characteristic of one of the newly discovered earthworms, Fridericia heliota, was the location of its luminescence: in the epidermal cells as opposed to coelomic fluid, typical for all bioluminescent oligochaetes reported earlier.52 It was established that bioluminescent system of F. heliota comprises five components: luciferin, luciferase, molecular oxygen, ATP, and Mg2+ ions, making it similar to that of fireflies.54,55

cell−cell interaction studies and for bioimaging in vivo of tumors,14,31 bacterial32 and other infections (malaria,33 fungi16), and so forth.34,35

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NEWLY IDENTIFIED BIOLUMINESCENT SYSTEMS

For over 25 years, considerable effort of various researchers from academic and commercial fields alike was aimed at improvement and adaptation of known bioluminescent systems for the evergrowing range of applications in biomedical research. There is no perfect luciferase or luciferin for every analytical method. For example, firefly luciferase is ATP-dependent and could be inhibited by certain intercellular factors,36 and Metridia longa luciferase is inactivated by blood serum albumin.37 Opposite to firefly luciferase, secreted Cypridina, Gaussia, and Metridia luciferases have their own advantages, such as the light signal independency on the substrate concentration within the cell. Small NanoLuc luciferase (19 kDa)38 is very useful in viral bioimaging research, as viral genome carrying long reporter gene becomes unstable.39−41 Finally, luciferins and their analogues differ in stability, cell permeability, solubility in water, and quantum yields of bioluminescence.1 Usually, red-shifted emission, which is required for deep-tissue bioimaging because of absorption and scattering of light by tissues,42 may be achieved by developing synthetic luciferin analogues,43−45 engineered luciferases,46 or BRET pairs,47−49 or by discovery of new bioluminescent systems. In the past few years, two such systems were unraveled: those of the earthworms Fridericia heliota and the higher fungi. Both newly discovered bioluminescent systems proved to be of a luciferase type, as it was possible to separate enzyme and substrate fractions of the biomass extract.50,51 In both cases, the individual components were inactive, but luminescence activity was regained by the recombination of the fractions in the presence of oxygen.

CompX, AsLn2: NMR Studies

As cultivation of Fridericia heliota worms under laboratory conditions proved to be impossible, the accumulation of earthworm biomass became the limiting factor in the investigation of its bioluminescent system. Due to the minute amount of Fridericia heliota luciferin (5 μg) obtained from the substrate fraction of earthworm biomass extract, it was impossible to directly establish its chemical structure.50 Therefore, we performed preliminary studies with two most abundant compounds present in the substrate fraction which had similar chromatographic and spectral behavior to those of luciferin. 2374

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Accounts of Chemical Research These compounds were termed CompX56 and AsLn257 (Figure 2).

The identification of Fridericia luciferin structure raised new questions. The close similarity of the luciferin fluorescence emission spectrum to the bioluminescence spectrum of Fridericia heliota (λmax 466 and 480 nm, respectively)56 implied that CompX moiety plays the role of light emitter, which meant that CompX conjugated π-system remains unchanged during bioluminescence reaction. In that case, the part of the molecule that undergoes oxidation, and consequently the mechanism of Fridericia bioluminescence, continued to be a mystery. Oxyluciferin, Novel Mechanism of Bioluminescence

In order to provide the structural basis for understanding the new mechanism of light emission underlying Fridericia bioluminescence, the elucidation of luciferin oxidation product (Figure 4,

Figure 2. Structures of compounds present in the substrate fraction of F. heliota biomass extract.

The structures of CompX and AsLn2 were established by analysis of their 1D and 2D NMR spectra, HRMS data and confirmed by total synthesis.56,58 We found CompX to be an unprecedented tyrosine analogue likely resulting from deamination, O-methylation, and aromatic carboxylation of tyrosine. In turn, a thorough examination of AsLn2 showed it to be a derivative of CompX, whose carboxyl groups are attached through peptide bonds to lysine and tyrosine residues. Structure Elucidation of Fridericia Luciferin

Figure 4. Structure of oxyluciferin.

The proton NMR spectrum of F. heliota luciferin revealed a pattern of signals in the aromatic region similar to those of CompX.56 Further NMR and HRMS experiments helped us to reveal three additional fragments of F. heliota luciferin. Apart from CompX moiety, the luciferin structure consisted of lysine, γ-aminobutyric acid (GABA), and monosubstituted oxalic acid residues.50 Four isomeric structures differing only in the order of the peptide bonds connecting the four residues conformed to the resulting spectral data. We synthesized all four isomers and compared their NMR spectra to those obtained for the natural luciferin. Only one of the synthetic compounds (Figure 3,

oxyluciferin) structure became an essential task. We prepared oxyluciferin by mixing synthetic Fridericia luciferin with excess ATP and crude Fridericia luciferase, obtained through partial purification of the enzyme extract.59 The collected NMR and HRMS data allowed us to conclude that Fridericia oxyluciferin is produced through oxidative decarboxylation of the lysine moiety (Figure 4).60 This result provided unambiguous evidence that oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as a light emitter. The multistage mechanism of Fridericia bioluminescence seems to be highly similar to that of fireflies, both of which begin with the formation of luciferyl adenylate (Scheme 3). Subsequent deprotonation of lysine α-CH and addition of O2 via single electron-transfer (SET) oxidation, as proposed by Mofford et al.61 and Branchini et al.62 for fireflies, leads to the formation of dioxetanone intermediate. In the final stage the release of CO2 generates oxyluciferin in an electronically excited state, which produces blue light upon relaxation to the ground state. If the reaction proceeds according to the proposed mechanism, then luciferyl adenylate should be the intermediate product of F. heliota bioluminescence. In order to demonstrate bioluminescent properties of the adenylate we tried to perform its chemical synthesis in several attempts, all of which proved to be unsuccessful. Therefore, a chemiluminescence study of a model compound bearing tert-butyl substituent at the lysine carboxy group (Figure 5) under the action of bases was implemented. The observed luminescence confirmed that the formation of ester at the lysine carboxyl group increases the αCH-acidity of this compound making it susceptible to deprotonation and further oxidation to form peroxide intermediate. Thus, the bright chemiluminescence of a model compound has confirmed our hypothesis about the reaction mechanism of F. heliota bioluminescence, in which the luciferase plays a dual role: initially catalyzing the adenylation reaction with Mg-ATP and then acting as a deprotonation agent.

Figure 3. Four possible luciferin structures. Compound 1, true Fridericia luciferin.

luciferin 1) was found to be identical to the natural substrate of F. heliota bioluminescent system, and produced light when introduced into F. heliota luciferase assay. The novel luciferin was found to have an unusual extensively modified peptidic nature, thus implying an unprecedented ATP-dependent bioluminescence mechanism. 2375

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Accounts of Chemical Research Scheme 3. Luciferase-Catalyzed Mechanism of Fridericia Bioluminescence

synthesis and NMR studies of natural and synthetic peptides proved that all discovered natural analogs of Fridericia luciferin (AsLn5, AsLn7, AsLn11, and AsLn12) possess Z-configuration of double bond in the modified tyrosine moiety. The structures of luciferin and its analogues AsLn5, AsLn7, AsLn11, and AsLn12 represent a very unusual biochemistry for terrestrial animals. The abundance of analogs in Fridericia biomass and the similarities between their chemical structures with that of luciferin allows us to speculate on the biosynthetic pathway of Fridericia luciferin. As AsLn7 contains the same modified tyrosine dicarboxylate core (CompX) and γ-aminobutyric acid side chain as found in the luciferin, it could be reasonable to assume that CompX moiety is consecutively bound to amino acids (GABA and lysine) and oxalic acid as it progresses through luciferin biosynthetic pathway (Figure 7). The other three structures (AsLn5, AsLn11, and AsLn12) comprise a different modified tyrosine core structure (CompY) that lacks the carboxylic acid group at ortho-position to the phenolic hydroxyl present in CompX. We have found that

Figure 5. Structure of a chemiluminescent model compound for luciferin adenylate.

Natural Luciferin Analogues: Novel Peptide Chemistry

Apart from two major compounds used as models in luciferin studies a number of minor highly unusual peptides were discovered in the substrate fraction of Fridericia biomass extract.63 These natural compounds contained either of the two tyrosine-derived chromophores, CompX or CompY (see below), and various (modified) amino acids (Figure 6). Total

Figure 6. Peptides from Fridericia heliota comprising either of the two tyrosine-derived chromophores: CompX (AsLn7) or CompY (AsLns5, AsLn11, AsLn12). 2376

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luminescence were made in the late 1950s and early 60s;68,69 however, despite a great interest toward this subject in the past 50 years, until quite recently its biochemical basis remained unknown. In their works, Airth and Foerster have proposed that fungal bioluminescence reaction is a two-stage process involving a NAD(P)H-dependent soluble enzyme and a membrane-bound insoluble luciferase.69 The first stage is a dark reaction catalyzed by a soluble enzyme, in which NAD(P)H plays a role of electron donor for an electron-accepting luciferin precursor yielding luciferin. At the next luciferase-catalyzed stage, true luciferin is oxidized by molecular oxygen resulting in light emission (Scheme 4). Scheme 4. Two-Stage Fungal Bioluminescence Mechanism Proposed in 1962 Figure 7. Two possible pathways of Fridericia luciferin biosynthesis.

instead of modified lysine attached through a peptide bond found in luciferin, the 3 new compounds have threonine (AsLn5), homoarginine (AsLn12) or an N,N-dimethylarginine (AsLn11) residues appended. Although CompY was not isolated from the earthworm biomass, the presence of AsLn12 in the extract suggests alternative Fridericia luciferin biosynthetic pathway proceeding through replacement of guanidinium fragment in homoarginine by oxalic acid residue. While the finding of AsLn7 favors CompX as a luciferin precursor, both proposed biosynthetic pathways deserve consideration. As a result of this decade-long exploration of a novel earthworm bioluminescent system, several unexpected discorevies were made. It was established that Fridericia luciferin is of unusual peptidic nature, placing it in the same class with marine luciferins: coelenterazine and Cypridina luciferin. It might also be noted that firefly luciferin could conceivably also fall into this class, as it is derived from two cysteine residues.64 ATPdependence of Fridericia bioluminescence reaction makes it similar to that of fireflies and beetles and only second of this type among all fully characterized bioluminescent systems. It is also worth noting that the chemical structure and the unprecedented mode of action of novel luciferin provide opportunity for creation of functionally active synthetic analogs of Fridericia luciferin with red-shifted bioluminescence emission spectra, owing to the fact that the structural fragments responsible for oxidation and light emission are separated in space, as opposed to other luciferins (see the Supporting Information, for example). Also, higher chemical stability of Fridericia luciferin (stable on air in solution for unlimited time, unusual peptidic substrate cannot be recognized by proteases and cleaved)65 compared to known luciferins makes it a promising candidate for using in the vast diversity of biotechnological applications based on bioluminescence, as soon as Fridericia luciferase is identified and cloned.

Hispidin: “Luc, I Am Your Father”

In our recent work the structures of fungal luciferin and its precursor were identified for the first time.51 The fungal luciferin did not present structural similarity to any of the eight luciferins discovered previously allowing us to describe a novel chemical basis of bioluminescence. In early 2015, we found that fruiting bodies of nonluminous fungal species Pholiota squarrosa contain luciferin precursor in quantities a hundredfold greater than those in glowing fungi.51 Soaking of the bioluminescent fungal mycelium in distilled water overnight surprisingly led to a great increase in its concentration as well. As became evident from NMR and HRMS data luciferin precursor turned out to be a member of a widespread styrylpyrone class of fungal and plant secondary metabolites, hispidin.70 Cross-reaction experiments showed this substrate to be ubiquitous in bioluminescence reactions of different species of luminous fungi. Further work allowed us to enzymatically convert hispidin into fungal luciferin using NADPH-dependent soluble enzyme fraction from luminescent fungi Neonothopanus nambi and determine its structure (Scheme 5). Scheme 5. Enzymatic Synthesis of Fungal Luciferin from Hispidin

Fungal Luciferin

Fungal luciferin was identified as 3-hydroxyhispidin, an oxidation product of the precursor hispidin. Its structure was confirmed by synthesis.71 When mixed with the luciferase fraction of N. nambi, the new compound produced a bright dose-dependent, NADPH-independent luminescence, which confirmed earlier postulated hypothesis on involvement of two separate enzymes in the fungal bioluminescence reaction (Scheme 4), thus confirming the functional role of this compound as the fungal luciferin. The luciferin−luciferase reaction in higher fungi does not require any additional cofactors except oxygen. Our data indicate that hydroxylation of hispidin at the pyranone fragment represents the first step in the sequence of

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DISCOVERY OF FUNGAL LUCIFERIN Unlike the radiant Fridericia earthworms, that were discovered only a decade ago, the earliest records of bioluminescent fungi date back two millennia.1 Since antiquity approximately 70 species of glowing fungi distributed over 9 genera were found.66 All reported luminescent fungi emit green light with maximum intensity within the range of 520−530 nm,5 and are likely to share a single bioluminescent system.67 The first documented academic inquiries into the phenomenon of fungal bio2377

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Accounts of Chemical Research chemical transformations, leading to fungal bioluminescence. The identification of a novel fungal luciferin and its biosynthetic precursor provides the chemical basis for a phenomenon that was observed millennia ago and opens up new horizons in applications, including the yet unmet challenge of creating autonomously luminescent plants.72

Chemistry of the Russian Academy of Sciences in 2015 with Ilia V. Yampolsky, where her principal research was synthesis of Fridericia luciferin. Presently, she occupies a postdoc position at the same Institute. Zinaida M. Kaskova was born in 1990 and raised in Moscow, Russia. She received her M.S. in chemistry from the Higher Chemical College RAS (Moscow) and her Ph.D. in bioorganic chemistry from the Institute of Bioorganic Chemistry of the Russian Academy of Sciences in 2016 with Ilia V. Yampolsky, where her principal research was synthesis of fungal luciferin and its analogues. Presently, she occupies a postdoc position at the same Institute.

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PROBLEMS IN PROGRESS As bioluminescence reactions involve two major components, enzyme and substrate, required for light emission, to attain full understanding of any bioluminescent system in its entirety, it becomes necessary not only to determine the structure of luciferin, but to characterize the macromolecule luciferase as well. Our present research is focused on identification and sequencing of Fridericia and fungal luciferases. Luciferases of new bioluminescent systems are not homologous to the known luciferases; therefore, the best strategy is targeted to obtain a genomic library for further expression in bacteria and yeast, and separate clones could be tested in an assay with the corresponding luciferin. However, as the luciferase transcripts may have low concentration, certain difficulties may occur, associated with low or absent expression, misfolding, or extensive post-translational modification of the expressed protein (leading to low bioluminescent activity). Another strategy is aimed at purification of the target protein in native conditions. The main limitations are low concentration of the protein and loss of the activity upon purification. Preliminary results show that both Fridericia and fungal luciferase may be purified in an active state using a combination of protein separation techniques including orthogonal chromatographies and gel electrophoresis (see the Supporting Information for more information). Further efforts of our group will be focused on the investigation of the amide ligases, responsible for biosynthesis of Fridericia luciferin-like modified peptides of CompX and CompY. We hope this approach will give us a better insight into the biochemistry of Fridericia luciferin biosynthetic enzymes and intermediates. As for the fungal bioluminescent system, the isolation and characterization of fungal oxyluciferin and 18O2labeling experiments with luciferin will hopefully shed light on the mechanism of its light emission.

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Ilia V. Yampolsky was born in 1979 and raised in Moscow, Russia. He received his M.S. in chemistry from the Higher Chemical College (Moscow) and his Ph.D. in bioorganic chemistry from the Institute of Bioorganic Chemistry of the Russian Academy of Sciences in 2009 with Sergey Lukyanov, where his principal research was structure elucidation of the chromophores of GFP-like red fluorescent proteins. Presently, he occupies a position of laboratory head at the same Institute.

ACKNOWLEDGMENTS

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REFERENCES

The authors gratefully acknowledge the support provided by the Russian Science Foundation Grant 16-14-00052.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.accounts.6b00322. Structures of known light-emitters in bioluminescent reactions, synthesis of Fridericia heliota luciferin analogues and their spectral properties, preliminary results on Fridericia and fungal luciferase purification (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Aleksandra S. Tsarkova was born in 1986 and raised in Moscow, Russia. She received her M.S. in chemistry from RUDN University (Moscow) and her Ph.D. in bioorganic chemistry from the Institute of Bioorganic 2378

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