Valentin N. Petushkov

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

A Novel Type of Luciferin from the Siberian Luminous Earthworm Fridericia heliota: Structure Elucidation by Spectral Studies and Total Synthesis

The structure elucidation and synthesis of the luciferin from the recently discovered luminous earthworm Fridericia heliota is reported. This luciferin is a key component of a novel ATP-dependent bioluminescence system. UV, fluorescence, NMR, and HRMS spectroscopy studies were performed on 0.005 mg of the isolated substance and revealed four isomeric structures that conform to spectral data. These isomers were chemically synthesized and one of them was found to produce light when reacted with a protein extract from F. heliota. The novel luciferin was found to have an unusual extensively modified peptidic nature, thus implying an unprecedented mechanism of action.

Redescription of Fridericia heliota (Annelida, Clitellata: Enchytraeidae), a luminous worm from the Siberian taiga, with a review of bioluminescence in the Oligochaeta

The Siberian enchytraeid Fridericia heliota Zalesskaja, 1990 has so far been known only through a paper in Russian briefly describing its luminescence and the major external and internal morphological features. The present study expands that preliminary description, amending it as regards the chaetal formula, the clitellar pattern, the form of the nephridia and the occurrence of glands at the spermathecal orifices, and providing a more comprehensive taxonomic characterization of the species. More detailed information is also given on its habitat and geographical range as well as on its unusual luminous properties. In F. heliota, the production of light is confined to the body wall with a pattern corresponding to that of the epidermal gland cells, although it does not involve discharge of luminous mucus. This contrasts with observations on other enchytraeids and on the oligochaetes in general. Records of luminosity in the Oligochaeta are reviewed with comments.

ATP is a cosubstrate of the luciferase of the earthworm Fridericia heliota (Annelida: Clitellata: Oligochaeta: Enchytraeidae).

The luminescence system of the soil enchytreid Fridericia heliota (this species has been discovered in 1990 [1]) is peroxide-independent and belongs to the luciferin–luciferase type [2]. Certain general properties of this luminescence system were studied both in vivo and in vitro. It was found that the reaction of light emission includes at least four basic components: an enzyme (luciferase), a reaction substrate (luciferin), magnesium ions, and oxygen [2]. According to preliminary data, the native molecular weight of the F. heliota luciferase is about 40 kDa. Because the activity of the enzyme rapidly declines during purification, the pure preparation of the F. heliota luciferase has not yet been obtained. Although the thermal stability of the luciferase preparation obtained by alkaline (or acid) extraction from worms was high, the ability of the resultant preparation to initiate luminescence was lost during long-term storage. The goal of this work was to study the ATP-dependent luminescence of F. heliota and to elucidate specific properties of the reaction substrate. It was shown in the preceding work [2] that the addition of adenosine phosphates (ATP, ADP, and AMP) to reaction mixture containing the preparations of luciferase and luciferin caused inhibition of luminescence. It was suggested that the effect of inhibition observed in these experiments was due to chelation of magnesium ions by phosphate groups, because magnesium ions contained in preparations were involved in the reaction of light emission. A detailed study of this effect gave rise to a completely new insight into the luminescence system of F. heliota. The final concentration of adenosine phosphates in reaction mixture was more than 30 times higher than the concentrations of magnesium ions the (concentration of magnesium ions in preparations of luciferase and luciferin, determined by the method of atomic absorption analysis, were similar and equaled to approximately 2.8 × 10 –4 M). The addition of an excess of magnesium ions should restore the luminescence intensity to the initial level. However, it was found that the intensity of the light emission was not only restored to the initial level but even exceeded this level by many times (Fig. 1, curve a ). A repeated addition of ATP caused no changes in the luminescence kinetics, which was indicative of saturating concentration of ATP in reaction mixture. Therefore, the decrease in the luminescence intensity was caused by other factors.

Study of the luminescence system of the soil enchytraeid Fridericia heliota (Annelida: Clitellata: Oligochaeta: Enchytraeidae).

Luminescent earthworms were first observed in Russia almost 200 years ago. In 1838, luminescent oligochaetes, identified as a new species Lumbricus noctilicus , were found in Kazan [1]. They emitted weak luminescence distributed over the body, with a greater intensity at the ends, and left luminescent mucus on the fingers of the observer. Later, upon more thorough studying, these worms were classified with the earlier described species Enchytraeus albidus (Henle, 1837) [2]. In 1908, the occurrence of luminescent soil oligochaets in the Kaluga guberniya (region) and in Perm’ were reported. The worms discovered were identified as Henlea ventriculosa . It is interesting that, in the same work, the name Enchytraeus albidus was used as a synonym of Henlea ventriculosa [3]. The luminescence of these oligochaetes was their intrinsic property, not related to a possible contamination with luminescent bacteria [4]. Thereafter, for almost 80 years, luminescent earthworms were not observed in Russia.

Novel Mechanism of Bioluminescence: Oxidative Decarboxylation of a Moiety Adjacent to the Light Emitter of Fridericia Luciferin

A novel luciferin from a bioluminescent Siberian earthworm Fridericia heliota was recently described. In this study, the Fridericia oxyluciferin was isolated and its structure elucidated. The results provide insight into a novel bioluminescence mechanism in nature. Oxidative decarboxylation of a lysine fragment of the luciferin supplies energy for light generation, while a fluorescent CompX moiety remains intact and serves as the light emitter.

LC-MS and microscale NMR analysis of luciferin-related compounds from the bioluminescent earthworm Fridericia heliota

This paper presents the main results of RP-HPLC-MS and microscale NMR analysis performed on Accompanying similar to Luciferin (AsLn(x)), compounds present in extracts of the bioluminescent earthworm Fridericia heliota that display similarities with Fridericias luciferin, the substrate of the bioluminescent reaction. Three isomers of AsLn were discovered, AsLn(1), AsLn(2) and AsLn(3), all of which present a molecular weight of 529 Da. Their UV-Vis absorption spectra show maxima at 235 nm for AsLn(1), 238 and 295 nm for AsLn(2) and 241 and 295nm for AsLn(3). MS(n) fragmentation patterns suggest the existence of carboxylic acid and hydroxyl moieties, and possibly chemical groups found in other luciferins like pterin or benzothiazole. The major isomer, AsLn(2), presents an aromatic ring and alkene and alkyl moieties. These luciferin-like compounds can be used as models that could give further insights into the structure of this newly discovered luciferin.

Mechanism and color modulation of fungal bioluminescence

Study of fungal bioluminescence mechanisms generates development of a multicolor enzymatic chemiluminescence system. Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

Novel Peptide Chemistry in Terrestrial Animals: Natural Luciferin Analogues from the Bioluminescent Earthworm Fridericia heliota

We report isolation and structure elucidation of AsLn5, AsLn7, AsLn11 and AsLn12: novel luciferin analogs from the bioluminescent earthworm Fridericia heliota. They were found to be highly unusual modified peptides, comprising either of the two tyrosine-derived chromophores, CompX or CompY and a set of amino acids, including threonine, gamma-aminobutyric acid, homoarginine, and unsymmetrical N,N-dimethylarginine. These natural compounds represent a unique peptide chemistry found in terrestrial animals and rise novel questions concerning their biosynthetic origin.

Ca(2+)-activator of the luminescence system of the earthworms Henlea sp., (Annelida: Clitellata: Oligochaeta: Enchytraeidae).

The luminous earthworm species assigned by us to genus Henlea (provisional name, Henlea sp.) is capable of excreting luminous coelomic fluid. It was shown in the preceding work [1] that the luminescence system of this species of enchytreids included at least three structural components (luciferase, luciferin, and an activator), the reaction of light emission being observed only in the presence of oxygen. The molecular weight of the Henlea sp. luciferase is 72 kDa, and its molecule is a homodimer. It was also shown that the activator of the luminescence reaction was a thermally stable lowmolecular-weight (< 2 kDa) compound of nonprotein structure [1].