Biosynthesis of the Fairy Chemicals, 2-Azahypoxanthine and Imidazole-4-carboxamide, in the Fairy Ring-Forming Fungus Lepista sordida

Akinobu Ito, Jae-Hoon Choi, Hirohide Takemura, Mihaya Kotajima, Jing Wu, Shinji Tokuyama,

Article

Biosynthesis of the Fairy Chemicals, 2‑Azahypoxanthine and

Imidazole-4-carboxamide, in the Fairy Ring-Forming Fungus Lepista

sordida

Hirofumi Hirai, Tomohiro Asakawa, Hitoshi Ouchi, Makoto Inai, Toshiyuki Kan,

and Hirokazu Kawagishi *

Cite This: https://dx.doi.org/10.1021/acs.jnatprod.0c00394

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sı Supporting Information

ABSTRACT: Fairy rings resulting from a fungus−plant inter-

action appear worldwide. 2-Azahypoxanthine (AHX) and

imidazole-4-carboxamide (ICA) were ﬁrst isolated from the

culture broth of one of the fairy ring-forming fungi, Lepista

sordida. Afterward, a common metabolite of AHX in plants, 2-aza-

-oxohypoxanthine (AOH), was found in AHX-treated rice. The

biosynthetic pathway of the three compounds that are named as

fairy chemicals (FCs) in plants has been partially elucidated;

however, that in mushrooms remains unknown. In this study, it

was revealed that the carbon skeletons of AHX and ICA were

constructed from Gly in L. sordida mycelia and the fungus

metabolized 5-aminoimidazole-4-carboxamide (AICA) to both of

the compounds. These results indicated that FCs were

biosynthesized by a diversion of the purine metabolic pathway in L. sordida mycelia, similar to that in plants. Furthermore, we

showed that recombinant adenine phosphoribosyltransferase (APRT) catalyzed reversible interconversion not only between 5-

aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5′-monophosphate (AICAR) and AICA but also between ICA-ribotide (ICAR)

and ICA. Furthermore, the presence of ICAR in L. sordida mycelia was proven for the ﬁrst time by LC-MS/MS detection, and this

study provided the ﬁrst report that there was a novel metabolic pathway of ICA in which its ribotide was an intermediate in the

fungus.

he rings, ribbons, or arcs of fungus-stimulated and/or

fungus-suppressed plant growth, which often occur on

L. sordida is widespread in northern temperate zones

throughout the world and is one of the famous fairy ring-

forming fungi in Japan; thus this fungus was chosen as a

ﬂoors of woodlands and agricultural grasslands, are generally

called “fairy rings”. The term “fairy rings” derives from the

material for our chemical research. In 2010, two plant growth-

myths and superstitions associated with this phenomenon in

the Middle Ages. The fruiting bodies of fungi sometimes

appear on the fairy rings, and it has been reported that about

regulating substances, 2-azahypoxanthine (AHX) and imida-

zole-4-carboxamide (ICA), were isolated from the culture

broth of L. sordida mycelia, and this was the ﬁrst reported

,10

isolation of both of the compounds from a natural source.

AHX elongated the shoots and roots of bentgrass seedlings,

0 kinds of mushroom-forming fungi form fairy rings all over

the world. Since the ﬁrst scientiﬁc paper on fairy rings was

published in 1675 and the subsequent research about them was

reviewed in Nature in 1884, the real identity of the “fairy” had

,10

whereas ICA suppressed them dose-dependently.

After-

ward, a metabolite of AHX common in plants, 2-aza-8-

oxohypoxanthine (AOH), was found, and AOH also exhibited

growth-regulating activity against the bentgrass seedlings

been unknown before our research. The accepted notion of

growth stimulation for fairy rings is that the fungal mycelia

decompose the protein in the soil and the plant growth is

promoted by absorption of accumulated nitrite and nitrate in

Received: April 7, 2020

−7

the soil.

However, we questioned this notion and proposed

the possibility that fairy ring-forming fungi produced speciﬁc

plant growth regulators, and we tried to isolate the causative

chemicals from one of the fairy ring-forming fungi, Lepista

sordida.

XXXX American Chemical Society and

American Society of Pharmacognosy

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 1. (A) Chemical synthetic route from AICA to AHX and ICA and (B) purine metabolic pathway common in plants, animals, and

microorganisms. The two consecutive arrows in (B) represent a multistage enzyme reaction. The black frame represents the novel purine metabolic

pathway producing FCs in plants. FAICAR, N-formyl-AICAR; XMP, xanthosine monophosphate.

similar to AHX. The three compounds were named “fairy

chemicals” (FCs) after the title of the article in Nature in 2014

the purine biosynthesis but also for the biosynthesis of

pyrimidine, pyridine, histidine, and tryptophan.

that covered our research. FCs exhibited growth-regulating

activity not only against bentgrass but also against various

kinds of plants regardless of the families they belong to, and it

was also elucidated that FCs were endogenously produced in

Adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) is

the key enzyme in the direct recycling of free adenine into

purine nucleotide pools and catalyzes the Mg -dependent

22,25−33

reversible interconversion between adenine and AMP.

−11,13,14

In APRT-deﬁcient human cells, it is known that adenine is

converted into a poorly soluble derivative, 2,8-dihydroxyade-

nine (DHA), and the accumulation of DHA leads to

plants.

Furthermore, yields of rice grains, wheat grains,

and other crops were signiﬁcantly increased and plants

developed tolerances against cold, hot, and salt stress by

treatment with FCs, suggesting the possibility of their practical

urolithiasis and renal failure. APRT also recognizes other

substances having the purine-like skeleton such as hypoxan-

,10,15,16

use in agriculture.

As mentioned above, FCs are natural

thine and AICA as substrates. In the previous research, a

precursor incubation experiment that added AICAR into an L.

sordida mycelial culture was performed, and the accumulation

compounds with a variety of beneﬁcial biological activities;

however, it is little known about how they are biosynthesized

in fungi, especially in the fairy ring-forming fungi.

FCs are chemically synthesized from a common precursor,

-aminoimidazole-4-carboxamide (AICA) (Figure 1A).

Previous studies about FCs biosynthesis in plants proved that

there was a biosynthetic pathway of FCs similar to the

chemical synthetic one and FCs were biosynthesized from

of AHX was observed after supplementation. In addition, it

was also reported that the expression level of the APRT gene

17−20

was stimulated by addition of AICAR into L. sordida.

Herein, we ﬁrst examined whether FCs endogenously

existed in various mushrooms or not. Subsequently, we tried

to elucidate the biosynthetic pathway to AHX and ICA in L.

sordida mycelia which are able to produce large amounts of

both of the compounds. In order to elucidate the relationship

between AHX and ICA biosynthesis and the purine metabolic

11,13,14

AICA from the purine metabolic pathway (Figure 1B).

Furthermore, it was also reported that AHX was biosynthe-

sized from 5-aminoimidazole-4-carboxamide-1-β-D-ribofurano-

syl 5′-monophosphate (AICAR), which is a precursor of

fundamental purine metabolites such as adenosine mono-

phosphate (AMP), inosine monophosphate (IMP), and

hypoxanthine in the purine metabolic pathway in L. sordida

mycelia, and AHX biosynthesis was transcriptionally controlled

pathway in L. sordida mycelia, a labeling experiment using C-

double-labeled Gly ([1,2- C ]Gly) was performed. Further-

more, the fungus was incubated with C-double-labeled AICA

to conﬁrm whether AICA was the precursor of AHX and ICA.

Finally, an APRT gene was cloned from L. sordida mycelia, the

gene was overexpressed in Escherichia coli, and the enzyme

activity of recombinant LsAPRT (rLsAPRT) was tested to

investigate the involvement of LsAPRT in the biosynthesis of

AHX and ICA.

by AICAR.

In most living organisms, purine nucleotides are biosynthe-

sized by a series of enzymatic reactions that result in the

formation of IMP, and IMP is further converted into AMP or

guanosine monophosphate to satisfy the purine nucleotide

22−24

requirements in growing cells.

Moreover, free purine

RESULTS AND DISCUSSION

■

bases can be directly recycled by a salvage pathway in the cells

and incorporated into purine nucleotide pools. The reaction is

catalyzed by phosphoribosyltransferases, a group of enzymes

that are responsible for the transfer of the phosphoribosyl

group from α-D-5-phosphoribosyl 1-pyrophosphate (PRPP)

onto a nitrogenous base to form an N-ribose monophosphate

In previous reviews, we introduced the preliminary result of the

endogenous existence of AHX and AOH in 11 species of

mushrooms (Cortinarius caperatus, Hypholoma sublateritium,

Lyophyllum decastes, Tricholoma flavoviens, Flammulina velu-

tipes, L. sordida, Lyophyllum connatum, Lepista nuda, Grifola

13,14

frondosa, Pholiota adiposa, and Lyophyllum shimeji).

Very

and pyrophosphate. The enzymes are necessary not only for

recently, we developed an exhaustive and eﬃcient method for

J. Nat. Prod. XXXX, XXX, XXX−XXX

respectively. Black arrows represent the signals of C from [1,2- C ]Gly.

Article

Figure 2. (A) ¹³C NMR spectra of AHX and (B) ICA derived from [1,2- C ]Gly in CD OD. ¹³C NMR spectra were recorded on a JEOL lambda-

00 spectrometer at 125 MHz. Top and bottom indicate the spectra of authentic standards and puriﬁed AHX and ICA derived from labeled Gly,

Figure 3. (A) LC-MS/MS spectra of ¹³C-double-labeled AHX, ICA, and AICA in the culture broth and (B) in the mycelia. The mycelia were

extracted after incubation with 0.2 mM unlabeled AICA or labeled AICA for 5 days. Labeled AHX and AICA were detected in the negative mode,

and labeled ICA was analyzed in the positive mode. LC-MS/MS chromatograms indicate multiple reaction monitoring (MRM) for [4,8- C ]AHX,

[

2,5- C ]ICA, and [2,5- C ]AICA, respectively.

detection and quantiﬁcation of FCs by LC-MS/MS. By this

method, ICA detection that had been almost impossible due to

its very low sensitivity has become possible. Therefore, we

analyzed the extracts of the mushrooms by this method. As a

result, the presence of AHX and ICA in various mushrooms

and the presence of AOH in only L. sordida and L. nuda were

proven for the ﬁrst time (Figures S1, S2, S3, Table S1).

Furthermore, all FCs in L. sordida mycelia were successfully

quantiﬁed (Figure S4). L. sordida mycelia produce large

amounts of AHX and do not have a plant-like xanthine oxidase

that converts AHX into AOH as judged from genome

and ICA were endogenously produced in various mushrooms;

however, L. sordida mycelia in particular produce high amounts

of both of the compounds and are cultured easily. Therefore,

the fungus was chosen for further investigation of the

biosynthetic pathway to the compounds.

As mentioned in the introduction, we have revealed that FCs

are biosynthesized from the purine metabolic pathway in

11,13,14

plants.

To investigate whether AHX and ICA are

produced by a similar biosynthetic pathway in L. sordida

mycelia, the fungus was incubated with [1,2- C ]Gly. It is

known that two juncture carbons in the purine carbon skeleton

of all the purine bases come from Gly in the purine metabolic

information. Therefore, AOH had not been detected from

L. sordida mycelia previously. This is the ﬁrst detection of

AOH from the fungus, although its content was very low. AHX

pathway. After incubation of the fungus with double-labeled

Gly, AHX and ICA were puriﬁed from the culture broth and

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 4. (A) UPLC analysis of the products of phosphoribosylation and (B) dephosphoribosylation using rLsAPRT. Puriﬁed rLsAPRT was

incubated with each substrate in the presence of Mg and PRPP or pyrophosphate at 37 °C for 12 h. Boiled enzyme was used for the negative

control. The reaction products were identiﬁed by comparing the retention time with that of authentic standards. t , retention time.

both of the compounds were analyzed by ¹³C NMR

spectroscopy and LC-MS/MS. In the ¹³C NMR spectra, the

signals of the juncture carbons, C-4 and C-5, in AHX and ICA

were remarkably increased and coupled to each other to form

doublets (Figure 2). In the LC-MS/MS analyses, unlabeled

AHX and ICA (authentic standards) gave only native parent

ion peaks (m /z 136 and 110, respectively) in the negative

mode (Figure S5A,B ). In contrast, puriﬁed AHX and ICA gave

parent ion peaks in which two carbons were derived from

labeled Gly (m/z 138 and 112, respectively) along with native

parent ion peaks (Figure S5C,D ). These results indicated that

two carbons of Gly were incorporated at the junctures of the

purine-like structure in AHX and ICA, and both compounds

were biosynthesized from the purine metabolic pathway in L.

sordida mycelia like the other general purine bases.

broth and the mycelial extract were analyzed by LC-MS/MS.

Labeled AHX and AICA were detected in the negative mode,

and labeled ICA was analyzed in the positive mode. As a result,

in the culture broth, labeled AHX and labeled ICA were

detected only in the labeled AICA treatment group and the

amounts of labeled AHX and ICA were 1.22 and 0.37 μmol,

respectively (Figure 3A). Residual labeled AICA in the

medium after the incubation of the fungus was not detected,

while the amount of labeled AICA in the medium without the

fungus was 1.97 μmol (Figure 3A), indicating that labeled

AICA was stable under this culture condition. In the mycelial

extract, the amounts of labeled AHX and ICA were very low

(12.8 and 9.0 nmol, respectively), and labeled AICA was not

detected (Figure 3B). Summarizing the result, the conversion

rates from AICA to AHX and to ICA were 62% and 19%,

respectively, and AICA was more used for the biosynthesis of

AHX than that of ICA in the fungus. AHX and ICA were for

the ﬁrst time isolated from the culture broth of the fungus, and

AICA has never been detected in the culture ﬂuid. The results

of the experiment suggested that most of labeled AICA was

absorbed into L. sordida mycelia, and then the fungus released

labeled AHX and ICA as metabolites of labeled AICA out of

the cells. This result clearly proved that there was a similar

biosynthetic route to AHX and ICA in the fungus to that in

plants. The reaction from AICA to AHX requires a nitrogen

source. According to the previous study, L. sordida mycelia

have a gene encoding nitric oxide (NO) synthase (NOS), and

In the chemical synthesis of AHX and ICA, AICA is the

17−20

precursor of both of the compounds.

AICA reacts with

NaNO₂to form an unstable intermediate, 4-diazo-4H-

imidazole-5-carboxamide (DICA), and treatment of DICA

with NH produces AHX (Figure 1A). ICA is also synthesized

by hydrogenolysis of DICA (Figure 1A). We thought that

AHX and ICA are biosynthesized in L. sordida mycelia by a

route similar to that of the chemical synthesis. In order to

examine whether AICA is the precursor of both of the

compounds in L. sordida mycelia or not, the fungus was

incubated with unlabeled AICA. As a result, the amounts of

AHX and ICA were signiﬁcantly increased by the treatment

Figure S6). Subsequently, ¹³C-double-labeled AICA, AHX,

NO is a candidate for the nitrogen source of the reaction.

(

and ICA were synthesized, and the fungus was incubated with

As mentioned above, AHX and ICA were endogenously

produced on the pathway as shown in Figure 1B not only in

plants but also in L. sordida mycelia. In the previous study, an

incubation experiment adding AICAR to a L. sordida mycelial

labeled AICA (2.0 μmol). An unlabeled AICA treatment

group and a labeled AICA treatment group without the fungus

were used as the controls in this experiment, and the culture

J. Nat. Prod. XXXX, XXX, XXX−XXX

Article

Figure 5. Detection of endogenous ICAR in L. sordida mycelia by LC-MS/MS. The freeze-dried mycelia were extracted and the sample was

analyzed by LC-MS/MS as described in the Experimental Section. ICAR was detected in the positive mode. LC-MS/MS chromatograms indicate

MRM for (A) authentic ICAR and (B) endogenous ICAR in the sample, respectively. t , retention time.

culture was performed, and consumption of AICAR and

accumulation of AHX were observed after supplementation.

Furthermore, quantitative reverse transcription-PCR analysis

amounts of AHX and ICA, AICA might be quickly

metabolized to AHX, ICA, and/or other derivatives, and

some compounds and/or proteins might help the dephosphor-

ibosylation activity of LsAPRT in vivo. More notably, rLsAPRT

recognized ICA but not AHX and AOH as a substrate, and

LC-MS/MS indicated the endogenous existence of ICAR in L.

sordida mycelia (Figure 5). The presence of ICAR in

organisms was veriﬁed by this result for the ﬁrst time. A

plant hormone, cytokinins, plays vital roles in the regulation of

multiple physiological processes in plants, such as cell

proliferation, diﬀerentiation, leaf senescence, and responses

demonstrated that the APRT gene of L. sordida mycelia

showed transcriptional enhancement after adding of AICAR.

Considering these results, we thought that LsAPRT was

involved in the reaction from AICAR to AICA; therefore, the

gene was overexpressed in E. coli. rLsAPRT puriﬁed by Ni

aﬃnity column chromatography gave a single band between 20

and 25 kDa in SDS-PAGE, in agreement with the molecular

mass, 22.1 kDa, calculated from its amino acid sequence

41−45

(

Figure S7 ). As a result, rLsAPRT (about 2 to 4 mg) was

to biotic and abiotic signals.

It was previously reported

puriﬁed from 1 L of transgenic E. coli cell culture and used in

the following enzyme assays.

that APRT1 in Arabidopsis catalyzed the conversion from

isopentenyladenine (iP) and trans-zeatin (trans-Z) to their

The phosphoribosylation activity of rLsAPRT toward

adenine, hypoxanthine, AICA, AHX, ICA, and AOH and its

dephosphoribosylation activity using AMP, IMP, AICAR, and

ICA-ribotide (ICAR) were tested. Boiled enzyme was used in

the negative control. Evaluation of the results was done using

ultraperformance liquid chromatography (UPLC) of the

reaction mixtures. rLsAPRT transformed the majority of

adenine, AICA, and ICA to their phosphoribosylated products

AMP, AICAR, and ICAR (Figure 4A). As shown in Figure

S8A, the phosphoribosylation conversion eﬃciency was the

highest for adenine. On the other hand, the activity with

hypoxanthine was very low, and no product was formed in the

reaction employing AHX or AOH as an acceptor (Figure 4A).

Furthermore, rLsAPRT also catalyzed the dephosphoribosyla-

tion and converted AMP, IMP, AICAR, and ICAR into their

corresponding free bases (Figure 4B). However, the activity of

the reverse reaction utilizing all the substrates was considerably

lower than the phosphoribosylation activity. The dephosphor-

ibosylation activity of the enzyme toward AICAR was stronger

than that for ICAR (Figure S8B). rLsAPRT exhibited the

highest activity at 40 °C and was stable at temperatures lower

than 50 °C (Figure S9A,B ). The enzyme showed the highest

activity at pH 5.0, and the activity remained more than 60%

between pH 5.0 and 10.0 (Figure S9C,D ).

nucleotides. Deactivation of APRT1 in the plant led to excess

accumulation of the two cytokinins, thus evoking various

cytokinin-regulated responses such as delayed leaf senescence,

anthocyanin accumulation, and downstream gene expression.

In addition, cytokinins derived from Ustilago maydis, the

causative principle of maize smut, play a key role in regulating

U. maydis and Zea mays interaction. The strain in which

biosynthesis of cytokinins was deﬁcient elicited fewer and

smaller tumors than wild type. iP and cis-zeatin (cis-Z) are

converted into their nucleotides by APRT in U. maydis, and

the infection to maize might be regulated by phosphoribosy-

lation of them. Therefore, the biological activities of FCs might

be regulated by phosphoribosylation and dephosphoribosyla-

tion in organisms like in the case of cytokinins. Furthermore,

genome sequence analysis of L. sordida mycelia showed that

the fungus has other genes encoding phosphoribosyltrans-

ferases. Generally, the substrate speciﬁcity of phosphoribo-

syltransferases such as APRT and hypoxanthine-guanine

phosphoribosyltransferase (HGPRT) is diﬀerent from each

other and the enzymes recognize diﬀerent compounds having a

48,49

purine-like skeleton as substrates.

Therefore, AHX and

AOH might be metabolized to their ribotides by other

phosphoribosyltransferases such as HGPRT in L. sordida

mycelia.

FCs are plant growth regulators isolated from the fairy ring-

forming fungus L. sordida as the causative principles of fairy

rings. In conclusion, we succeeded in detecting at least one

kind of FC in 11 species of mushrooms and partially

elucidating the biosynthetic pathway to AHX and ICA in L.

sordida mycelia. The most important ﬁndings in this study

were that rLsAPRT catalyzed the reversible interconversion

In this study, the function of rLsAPRT became clear in vitro

for the ﬁrst time. However, contrary to our expectation, the

conversion activity from AICAR to AICA was much lower than

that from AICA to AICAR. In the previous study, it was

reported that the phosphoribosylation activity toward adenine

of APRT derived from Leishmania donovani was more than 10

times higher than the dephosphoribosylation activity toward

AMP. Considering that L. sordida mycelia produce large

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

between ICA and ICAR, and the endogenous existence of

ICAR was veriﬁed in L. sordida.

0.0001% MnSO

·5H

O, 1% D-glucose, 0.05% KH

, 0.05%

Na HPO , pH 5.5), and the culture was incubated for 1 week (25

C, 120 rpm). Labeled AICA or unlabeled AICA ﬁltered through a

membrane ﬁlter was added to the culture, and the mycelia were

further cultured for 5 days (25 °C, 120 rpm).

EXPERIMENTAL SECTION

■

General Experimental Procedures. ¹³C NMR spectra were

recorded on a JEOL lambda-500 spectrometer at 125 MHz. LC-MS/

MS analyses were performed with a UPLC system (Shimadzu)

coupled with a tandem LTQ Orbitrap mass spectrometer (Thermo

Fisher Scientiﬁc) and a UPLC system (Nihon Waters) coupled with a

tandem Xevo TQ-S micro mass spectrometer (Nihon Waters). HPLC

separations were performed with a Jasco Gulliver system (PU-2089

Plus quaternary gradient pump, AS-2010 Plus multiwavelength

detector, AS-2055 Plus Intelligent sampler, CO-2060 Plus Intelligent

column thermostat, LC-Net II/ADC). The enzyme assays of

rLsAPRT were performed with a UPLC system (quaternary solvent

manager, sample manager-FTN, PDA eλ detector, Nihon Waters).

Isolation and Detection of Labeled AHX and Labeled ICA

Derived from [1,2- C ]Gly. The culture broth (1 L) of L. sordida

mycelia was ﬁltered and concentrated under reduced pressure. The

concentrate (5.1 g) was fractionated by octa-decyl-silyl gel (Cosmosil

(

40C18-OPN, Nacalai Tesque) ﬂash column chromatography: 5%

fractions 1 to 5), 10% (fractions 6 to 10), 25% (fractions 11 to 15),

and 50% (fractions 16 to 20) MeOH in H O and MeOH (fractions

21 to 25) to obtain 25 fractions (fractions 1 to 25). Fractions eluted

with 10% MeOH were combined, and the fraction (400.7 mg) was

separated by HPLC (column, Develosil C30-UG-5, ϕ 20 × 250 mm,

Nomura Chemical; solvent, 2% MeOH in 0.02% triﬂuoroacetic acid

(TFA); ﬂow rate, 5.0 mL/min; UV detection, 210 nm: column,

Cosmosil 5-PYE waters, ϕ 10 × 250 mm, Nacalai Tesque; solvent, 2%

MeOH in 0.02% TFA; ﬂow rate, 2.0 mL/min; UV detection, 210 nm)

to aﬀord AHX (4.9 mg) and ICA (3.5 mg). Puriﬁed AHX and ICA

were dissolved with 95% MeOH in 0.05% FA. The sample was

centrifuged, and then the supernatant was subjected to LC-MS/MS

analysis (Thermo Fisher Scientiﬁc). A PC-HILIC column was used in

this analysis (solvent, 95% MeOH in 0.05% FA; column oven, 40 °C;

ﬂow rate, 0.2 mL/min; injection volume, 10 μL). MS analysis

including ICA was performed in the negative mode with the same

source parameters above.

Stable-isotope-labeled Gly ([1,2- C ]Gly) and authentic AICAR

were purchased from Cambridge Isotope Laboratories and Sigma-

Aldrich Japan, respectively. C-double-labeled AICA, AHX, ICA, and

authentic ICAR were synthesized according to the methods

39,50

previously described.

All solvents used throughout the experi-

ments were obtained from Kanto Chemical.

Fungal Material. The fruiting bodies of Cortinarius caperatus,

Hypholoma sublateritium, Tricholoma flavoviens, Lepista sordida,

Lyophyllum connatum, Lepista nuda, and Pholiota adiposa were

collected at Narusawa village, Yamanashi Prefecture in Japan. The

fruiting bodies of Grifola frondosa, Flammulina velutipes, Lyophyllum

decastes, and Lyophyllum shimeji were purchased at local supermarkets

in Japan.

Detection of Endogenous FCs in Mushrooms. Lyophilized

mushrooms (about 50 mg) were used in this experiment. Extraction

and fractionation were performed as previously described. The

fraction was dissolved in 100 μL of 80% MeCN in 0.05% formic acid

Heterologous Expression of Recombinant LsAPRT in E. coli.

L. sordida mycelia were frozen in liquid nitrogen and ground into a

powder. Total RNA was extracted from the resulting mycelial powder

using TRIzol reagent (Thermo Fisher Scientiﬁc) and RNeasy mini

kit(Qiagen), according to the manufacturer’s instruction. cDNA was

reverse-transcribed from 500 ng of total RNA with Prime Script RT

reagent kit perfect real time (Takara Bio). A full-length cDNA

fragment of the LsAPRT gene was ampliﬁed by RT-PCR using

PrimeSTAR Max DNA polymerase (Takara Bio). A forward primer

(5′-gtgcatcatcatcatcatcatatcgaaggtaggcatATGGACGTTGAGTACAT-

TAAAGACCAATTGAC-3′) and a reverse primer (5′-

acaagcttgaattcgTCAATCATCCGATTGAACGATCG-3′) were de-

(

FA) and subjected to LC-MS/MS (Thermo Fisher Scientiﬁc). A PC-

HILIC column (ϕ 2.0 × 100 mm, Osaka Soda) was used in this

analysis (solvent, 95% MeCN in 0.05% FA; column oven, 40 °C; ﬂow

rate, 0.2 mL/min for ICA, 0.4 mL/min for AHX and AOH; injection

volume, 5 μL). For AHX and AOH detection, MS analysis was

performed in the negative mode with the following source parameters:

sheath gas ﬂow, 50; auxiliary gas ﬂow, 10; tube lens, −63 V; capillary

voltage, −16 V; ion spray voltage, 3 kV. For ICA detection, MS

analysis was performed in the positive mode with the following source

parameters: sheath gas ﬂow, 50; auxiliary gas ﬂow, 10; tube lens,

14.7 V; capillary voltage, 1 V; ion spray voltage, 3 kV. The

compounds were identiﬁed by exact mass and characteristic transition

precursor ion to daughter ion).

Lepista sordida Strain and Culture Conditions. The mycelia of

signed based on the nucleotide sequence of the LsAPRT gene.

The reaction mixture (10 μL) contained 50 ng of total cDNA, 5 μL of

PrimeSTAR Max DNA polymerase, and 0.25 μM of each primer.

Cycling conditions were set as follows: preincubation, 1 cycle of 94

°C for 2 min; ampliﬁcation, 35 cycles of 98 °C for 15 s, 60 °C for 30 s,

and 68 °C for 15 s. The ampliﬁed PCR products were analyzed by

electrophoresis using a 1.2% agarose gel and ligated into pCold I

expression vector (Takara Bio) between the NdeI and BamHI sites

using Gibson assembly master mix (New England BioLabs Japan),

followed by transformation into E. coli DH5α competent cells

(Nippon Gene). The plasmid DNA (pCold I-LsAPRT) was extracted

with the HiYield plasmid mini kit (RBC Bioscience). The authenticity

of the resulting plasmid DNA was conﬁrmed by PCR analysis and

sequencing, and then the plasmid was transformed into E. coli BL21-

CodonPlus (DE3)-RIPL competent cells (Agilent Technologies

Japan). The cells with the plasmid were grown in 1 L of lysogeny

broth (LB) medium containing 100 μg/mL ampicillin at 37 °C until

OD600 reached 0.4. After incubation at 4 °C for 30 min, rLsAPRT was

induced by 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG)

and the cells were cultured at 15 °C for at least 24 h. The cells were

harvested by centrifugation and stored at −80 °C until protein

extraction.

Puriﬁcation of rLsAPRT. The cells were suspended in extraction

buﬀer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 10% glycerol, and

30 mM imidazole). After lysis by sonication for 10 min on ice, the cell

debris was removed by centrifugation. The supernatant was ﬁltered

using a membrane ﬁlter and subjected to Ni aﬃnity column

chromatography using HisTrap HP (GE Healthcare Japan). After

washing the column with wash buﬀer (20 mM Tris-HCl, pH 7.4, 500

mM NaCl, 10% glycerol, and 70 mM imidazole), 6× His-tagged

rLsAPRT was eluted with elution buﬀer (20 mM Tris-HCl, pH 7.4,

−

(

L. sordida (NBRC112841) were precultured on potato dextrose agar

PDA) medium to obtain actively growing cultures, and the

(

inoculated mycelia were incubated at 25 °C for a month. After

growth, the mycelial plate was stored at 4 °C until it was used for the

experiments. For a labeling experiment using [1,2- C ]Gly, 30 pieces

(

6 mm diameter) cut from four-week-cultured mycelia on PDA

medium were placed into each 500 mL Erlenmeyer ﬂask containing

50 mL of YG medium (0.05% yeast extract and 0.5% D-glucose).

One milliliter of 62.5 mM [1,2- C ]Gly ﬁltered with a membrane

ﬁlter was added to the cultures, and the cultures were incubated for 5

weeks (25 °C, 120 rpm). For total RNA extraction and endogenous

FCs and ICAR detection in L. sordida mycelia, two pieces (8.5 mm

diameter) cut from a similar mycelial plate were inoculated into a 50

mL Erlenmeyer ﬂask containing 10 mL of YG medium (0.3% yeast

extract, 1% D-glucose, 0.05% KH PO , 0.05% Na HPO and pH 5.5),

and the culture was incubated for 3 weeks (25 °C, 120 rpm). The

mycelia were collected by ﬁltration and frozen at −80 °C until further

experiments. For a labeling experiment using C-double-labeled

AICA, 10 pieces (5.0 mm diameter) were inoculated into a 50 mL

Erlenmeyer ﬂask containing 10 mL of modiﬁed Czapek medium

(

0.3% NaNO , 0.1% K HPO , 0.05% MgSO ·7H O, 0.2% KCl,

3 2 4 4 2

.002% CuSO ·5H O, 0.001% FeSO ·7H O, 0.0001% ZnSO ·7H O,

4 2 4 2 4 2

J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

orcid.org/0000-0001-5782-4981 ; Phone: +81-54-238-

Article

00 mM NaCl, 10% glycerol, and 1 M imidazole) with a linear

AUTHOR INFORMATION

Corresponding Author

■

gradient (0.07 to 1 M imidazole). The recombinant protein eluted

fractions were conﬁrmed by 15% SDS-PAGE with Coomassie brilliant

blue staining and desalted against dialysis buﬀer (20 mM Tris-HCl,

pH 7.4, 500 mM NaCl, and 10% glycerol) at 4 °C. After dialysis, the

protein solution was concentrated and replaced with enzyme buﬀer

Hirokazu Kawagishi − Graduate School of Science and

Technology, Graduate School of Integrated Science and

Technology, and Research Institute of Green Science and

Technology, Shizuoka University, Shizuoka 422-8529, Japan;

4885; Email: kawagishi.hirokazu@shizuoka.ac.jp

(20 mM Tris-HCl, pH 7.4, 500 mM NaCl, and 20% glycerol) using

Amicon Ultra-0.5 mL 10K (Merck). The puriﬁed protein concen-

tration was quantiﬁed using the Pierce 660 nm protein assay reagent

(Thermo Fisher Scientiﬁc) with BSA as the standard.

In Vitro Enzyme Assays of rLsAPRT. For the phosphoribosy-

Authors

lation activity, an enzyme reaction mixture (100 μL) containing 0.2

mM substrate (adenine, hypoxanthine, AICA, AHX, ICA, or AOH), 1

Akinobu Ito − Graduate School of Science and Technology,

Shizuoka University, Shizuoka 422-8529, Japan; Research

Fellow of Japan Society for the Promotion of Science, Tokyo

mM PRPP (Sigma-Aldrich Japan), 10 mM MgCl ·6H O, 20 mM

Tris-HCl (pH 7.4), and 100 μg/mL rLsAPRT was prepared. For the

dephosphoribosylation activity, the enzyme reaction mixture (100

μL) consisted of 0.2 mM substrate (AMP, IMP, AICAR or ICAR), 1

mM sodium pyrophosphate decahydrate (Sigma-Aldrich Japan), 10

mM MgCl ·6H O, 20 mM Tris-HCl (pH 7.4), and 100 μg/mL

02-0083, Japan

Jae-Hoon Choi − Graduate School of Science and Technology,

Graduate School of Integrated Science and Technology, and

Research Institute of Green Science and Technology, Shizuoka

University, Shizuoka 422-8529, Japan

Hirohide Takemura − Graduate School of Science and

Technology, Shizuoka University, Shizuoka 422-8529, Japan;

Research Fellow of Japan Society for the Promotion of Science,

Tokyo 102-0083, Japan

Mihaya Kotajima − Graduate School of Science and Technology,

Shizuoka University, Shizuoka 422-8529, Japan

Jing Wu − Research Institute of Green Science and Technology,

Shizuoka University, Shizuoka 422-8529, Japan

Shinji Tokuyama − Graduate School of Integrated Science and

Technology, Shizuoka University, Shizuoka 422-8529, Japan

Hirofumi Hirai − Graduate School of Science and Technology,

Graduate School of Integrated Science and Technology, and

Research Institute of Green Science and Technology, Shizuoka

University, Shizuoka 422-8529, Japan

Tomohiro Asakawa − Marine Science and Technology, Tokai

University, Hiratsuka City, Kanagawa 259-1292, Japan

Hitoshi Ouchi − Department of Pharmaceutical Sciences,

University of Shizuoka, Shizuoka 422-8526, Japan

Makoto Inai − Department of Pharmaceutical Sciences,

University of Shizuoka, Shizuoka 422-8526, Japan;

orcid.org/0000-0001-7074-0520

rLsAPRT. Enzyme was boiled for 10 min before the reaction for use

as the negative control. The reaction mixture was preincubated at 37

°C for 5 min, and then the reaction was initiated by adding the

enzyme solution. The reaction was terminated by boiling the mixture

for 5 min, and then the mixture was centrifuged to precipitate

proteins. The optimum temperature and thermal stability of rLsAPRT

were tested in 20 mM Tris-HCl (pH 7.4) at diﬀerent temperatures (5

to 90 °C). The eﬀect of pH was determined in diﬀerent buﬀers (pH

.0 to 3.0, 20 mM Gly-HCl; pH 3.0 to 5.0, 20 mM sodium acetate−

acetic acid; pH 5.0 to 7.0, 20 mM PIPES-NaOH; pH 7.0 to 8.0, 20

mM Tris-HCl; pH 8.0 to 10.0, 20 mM Gly-NaOH) at 40 °C. The

enzyme reaction products in the supernatant were detected by UPLC

analysis (column, CAPCELL PAK ADME S3, ϕ 2.1 × 250 mm,

Osaka Soda; solvent, A: 10 mM ammonium formate, B: MeOH, 2%

solvent B or a linear gradient (20% to 100% solvent B) for adenine

and AMP; ﬂow rate, 0.2 mL/min; column oven, 40 °C).

Detection of Labeled AHX and Labeled ICA Derived from

C-Double-Labeled AICA and Endogenous ICAR. L. sordida

mycelia were freeze-dried and extracted according to the high-

sensitivity detection method for purine metabolites. The sample was

dissolved in 100 μL of H O with 0.05% FA and subjected to LC-MS/

MS (Nihon Waters). A CAPCELL PAK ADME S3 column was used

in this analysis (solvent, A: 10 mM ammonium formate, B: MeOH,

% solvent B; column oven, 40 °C; ﬂow rate, 0.2 mL/min; injection

Toshiyuki Kan − Department of Pharmaceutical Sciences,

University of Shizuoka, Shizuoka 422-8526, Japan;

orcid.org/0000-0002-9709-6365

volume, 1 μL). For AHX detection, MS analysis was performed in the

negative mode with the following source parameters: capillary voltage,

.5 kV; cone voltage, 40 V; collision energy, 12 V. For ICA detection,

MS analysis was performed in the positive mode: capillary voltage, 1.0

kV; cone voltage, 10 V; collision energy, 28 V. For AICA detection,

MS analysis was performed in the negative mode: capillary voltage,

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jnatprod.0c00394

.5 kV; cone voltage, 2 V; collision energy, 18 V. For ICAR detection,

Notes

MS analysis was performed in the positive mode: capillary voltage, 1.0

kV; cone voltage, 4 V; collision energy, 12 V. The following source

parameters were common to all the compounds: desolvation

temperature, 500 °C; desolvation gas ﬂow, 1000 L/h; cone gas

ﬂow, 50 L/h.

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Dr. K. Okamoto (Ushio Chemix, Shizuoka, Japan)

for providing authentic AHX and ICA. This work was partially

supported by a Grant-in Aid for Scientiﬁc Research on

Innovative Areas “Frontier Research on Chemical Communi-

cations” (JP17H06402) from MEXT to H.K. and a Japan

Society for the Promotion of Science KAKENHI (No.

ASSOCIATED CONTENT

■

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00394.

18J21506) to A.I.

LC-MS/MS spectra of FCs and detection of FCs in

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