Research Article: First evidence of cholinesterase-like activity in Basidiomycota

Date Published: April 30, 2019

Publisher: Public Library of Science

Author(s): Kristina Sepčić, Jerica Sabotič, Robin A. Ohm, Damjana Drobne, Anita Jemec Kokalj, Israel Silman.


Cholinesterases (ChE), the enzymes whose primary function is the hydrolysis of choline esters, are widely expressed throughout the nature. Although they have already been found in plants and microorganisms, including ascomycete fungi, this study is the first report of ChE-like activity in fungi of the phylum Basidiomycota. This activity was detected in almost a quarter of the 45 tested aqueous fungal extracts. The ability of these extracts to hydrolyse acetylthiocholine was about ten times stronger than the hydrolytic activity towards butyrylthiocholine and propionylthiocholine. In-gel detection of ChE-like activity with acetylthiocholine indicated a great variability in the characteristics of these enzymes which are not characterized as vertebrate-like based on (i) differences in inhibition by excess substrate, (ii) susceptibility to different vertebrate acetylcholinesterase and butyrylcholinesterase inhibitors, and (iii) a lack of orthologs using phylogenetic analysis. Limited inhibition by single inhibitors and multiple activity bands using in-gel detection indicate the presence of several ChE-like enzymes in these aqueous extracts. We also observed inhibitory activity of the same aqueous mushroom extracts against insect acetylcholinesterase in 10 of the 45 samples tested; activity was independent of the presence of ChE-like activity in extracts. Both ChE-like activities with different substrates and the ability of extracts to inhibit insect acetylcholinesterase were not restricted to any fungal family but were rather present across all included Basidiomycota families. This study can serve as a platform for further research regarding ChE activity in mushrooms.

Partial Text

Cholinesterases (ChEs), the enzymes that hydrolyse choline esters but also exert non-hydrolytic activities [1], are considered as one of the catalytically most efficient enzymes in nature [2]. Cholinesterases are also widely expressed in organisms from different taxonomic groups [3]. It has been reported that ChEs with highly selective substrate specificity had appeared in the early bilaterians [4]. Two qualitatively different ChEs, acetylcholinesterase (AChE; E.C. and butyrylcholinesterase (BChE; E.C., were characterised in vertebrates. Phylogenetic analysis of vertebrate BChE and AChE expression indicate that these two enzymes have emerged from a common precursor whose function was to hydrolyse acetylcholine [5]. In some invertebrates (e.g. in crustaceans) it has been suggested that ChEs show intermediary characteristics between the two vertebrate forms and can be classified as neither AChE nor BChE [6,7]. ChEs have been found also in organisms devoid of nervous system such as sponges (Karczmar, 2010), both Gram positive and Gram negative bacteria [8–15], ascomycete fungi [16–18], plants [19–22], and protozoa [23–26]. However, no studies so far have reported the ChE-like activities in fungi belonging to the phylum Basidiomycota.

To the best of our knowledge, higher fungi have not been a matter of ChE activity investigations up to now, and this study provides the first evidence of ChE-like activity in Basidiomycota using ACh, PCh and BCh as substrates. ChE activity was reported in several species of the phylum Ascomycota (genera Aspergillus, Penicillium and Fusarium) in the early ’90s [16–18]. However, the genes encoding these enzymes have not been identified. Interestingly, acetylcholine, a naturally occurring ChE substrate, has been detected in the extract of the ergot fungus, Claviceps purpurea, more than a hundred years ago [71].