The ninth formula of light

Anton Chugunov, IBH RAS 

The ghostly glow in the thicket of the night forest attracted the attention of people in ancient times. The first descriptions of this phenomenon are given in the works of Aristotle and Pliny. At the beginning of the XIX century, it turned out that the cause of the glow of wood is a fungal mycelium. Since then, a large list of different types of fungi that emit light at different stages of their development has accumulated. For more than a hundred years, scientists from different countries have been trying to solve the riddle of mushroom glow. However, time after time, all attempts to isolate the components involved in bioluminescence and to understand the mechanism of this process have failed. Nobel laureate Osamu Shimomura said this during his visit to Krasnoyarsk: "It's very hard to get results, it's hard work that takes a lot of time. In Japan and the United States, those who encounter such difficulties tend to find easier ways. I offered several times to conduct research on mushrooms in Japan, and I was refused. This is a very interesting topic, but too complicated. There are three types of bioluminescence in nature: the first is the luciferin-luciferase system, the second is the photobelk system, and the third... still not studied. Fundamentally new things are always difficult to do. I was primarily interested in Russian studies of the luminescence of fungi and worms. I believe those who study the glow of worms will be able to determine the structure of their luciferin, and I hope for significant progress in understanding the mechanisms of the glow of fungi."[1]. 

The same team that recently deciphered the luciferin Fridericia heliota, which became the "eighth formula of light", began to study glowing mushrooms [2]. As a result, the combined efforts of several laboratories (Laboratory of Photobiology and Laboratory of Nanobiotechnology and Bioluminescence of the Institute of Biophysics SB RAS (Krasnoyarsk), the Group of Synthesis of Natural Compounds of IBH RAS (Moscow)) a breakthrough was made in the topic of fungal bioluminescence. The structure of a new unique luciferin – 3-hydroxyhispidine and the path of its biosynthesis from the precursor were established, and data confirming a single biochemical mechanism of fungal bioluminescence were obtained. The results of the research are published in the journal Angewandte Chemie Int. Ed. [3]

How did scientists manage to isolate the "elusive" luciferin? The scheme of fungal bioluminescence assumes the formation of luciferin from a certain precursor. In the course of the work, it was found out that the luciferin precursor is also synthesized in non-luminous fungi, and in an amount 100 times greater than in luminous ones. Therefore, it was from non-luminous mushrooms that predluciferin was isolated. The use of a simple procedure for soaking the mycelium in water allowed to increase the concentration of pre-luciferin and the enzyme that converts it into luciferin by hundreds of times. And the destruction of fungal mycelium by ultrasound dramatically increased the efficiency of extracting the bioluminescent enzyme system into solution. The use of these techniques eventually led to success.

Using HPLC, 6 substances were obtained from Pholiota squarrosa mushroom extract, the structures of which were then determined by standard methods. The most active substance in bioluminescent tests turned out to be a well–known natural compound - hispidine. The Yuichi Oba group from Nagoya University (Japan) confirmed that hispidin is a universal precursor of luciferin in other fungi as well.

Fig. 1. Chromatogram of mushroom extract, structures of isolated substances and their luminescent activity.

Note an interesting fact that in 1998, in a personal message to Osamu Shimomura, his colleague Hideshi Nakamura suggested the content of a common fragment in the structure of the probable precursors of luciferin – caffeic acid, that is, he was one step away from discovering hispidin [4]. 

Further experiments showed that the enzyme converting hispidin to luciferin turned out to be not a reductase, as previously assumed, but a hydroxylase. By isolating this enzyme, it was possible to obtain the mushroom luciferin itself and then establish its structure. Thus, the scheme of fungal bioluminescence includes two substrates: preluciferin (hispidin) and luciferin (3-hydroxygispidin) - and two enzymes: hydroxylase converts preluciferin to luciferin, and luciferase oxidizes it with the emission of a quantum of light. 

Fig. 2. Scheme of fungal bioluminescence.

So the secret of mushroom luminescence was revealed, which excited the minds of many scientists. Osamu Shimomura's words turned out to be prophetic. A new bioluminescent system was described and mushroom luciferin became the ninth in the world list. Mushroom luciferin will still find its application in bioimaging and other areas of applied bioluminescence.

Literature:

13.07.2015