The coupled reaction catalyzed by EchB and EchC lead to the formation of the common 2′,3′,5′-trihydroxy-benzene core in echosides biosynthesis

Article abstract of DOI:10.1016/j.bbrc.2021.04.087

p-Terphenyls represent a unique family of aromatic natural products generated by nonribosomal peptide synthetase-like (NRPS-like) enzyme. After formation of p-terphenyl skeleton, tailoring modifications will give rise to structural diversity and various biological activities. Here we demonstrated a two-enzyme (EchB, a short-chain dehydrogenase/reductase (SDR), and EchC, a nuclear transport factor 2 (NTF2)-like dehydratase) participated transformation from dihydroxybenzoquinone core to 2′,3′,5′-trihydroxy-benzene in the biosynthesis of echosides. Beginning with polyporic acid as substrate, successive steps of reduction-dehydration-reduction cascade catalyzed by EchB-EchC-EchB were concluded after in vivo gene disruption and in vitro bioassay experiments. These findings demonstrated a conserved synthesis pathway of 2′,3′,5′-trihydroxy-p-terphenyls in bacteria, such as Actinomycetes and Burkholderia. The parallel pathway in fungi has yet to be explored.

Full text of DOI:10.1016/j.bbrc.2021.04.087

Biochemical and Biophysical Research Communications 559 (2021) 62e69
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The coupled reaction catalyzed by EchB and EchC lead to the
formation of the common 20,30,50-trihydroxy-benzene core in
echosides biosynthesis
Jing Zhu a, Mengyujie Liu b, Jingjing Deng b, Wang Chen b, Deyu Zhu c, Jing Duan a,
,
, b, **
Yaoyao Li b, Haoxin Wang a *, Yuemao Shen a
a State Key Laboratory of Microbial Technology, Shandong University, Qingdao, 266237, PR China
b Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, PR China
c Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, 250012, PR
China
a r t i c l e i n f o
a b s t r a c t
Article history:
p-Terphenyls represent a unique family of aromatic natural products generated by nonribosomal peptide
synthetase-like (NRPS-like) enzyme. After formation of p-terphenyl skeleton, tailoring modifications will
give rise to structural diversity and various biological activities. Here we demonstrated a two-enzyme
(EchB, a short-chain dehydrogenase/reductase (SDR), and EchC, a nuclear transport factor 2 (NTF2)-
like dehydratase) participated transformation from dihydroxybenzoquinone core to 20,30,50-trihydroxy-
benzene in the biosynthesis of echosides. Beginning with polyporic acid as substrate, successive steps of
reduction-dehydration-reduction cascade catalyzed by EchB-EchC-EchB were concluded after in vivo
gene disruption and in vitro bioassay experiments. These findings demonstrated a conserved synthesis
pathway of 20,30,50-trihydroxy-p-terphenyls in bacteria, such as Actinomycetes and Burkholderia. The
parallel pathway in fungi has yet to be explored.
Received 1 April 2021
Accepted 21 April 2021
Available online 28 April 2021
Keywords:
p-Terphenyls
Biosynthesis
Short-chain dehydrogenase/reductase (SDR)
NTF2-like dehydratase
© 2021 Elsevier Inc. All rights reserved.
1. Introduction
substituted quinones were catalyzed by a tridomain nonribosomal
peptide synthetase (NRPS)-like enzyme [4,5]. Although 20,30,50-
The 20,30,50-trihydroxy-p-terphenyls comprise a unique group of
natural products found in fungi, bacteria and lichens (Fig. 1). A broad
spectrum of biological activity has been reported for these mole-
cules, including cytotoxic, antibacterial, anti-inflammatory, antioxi-
dant, immunosuppressive, neuroprotective, antithrombotic, and
anticoagulant activities [1e3]. It has been established that p-ter-
trihydroxy-p-terphenyls are widespread in various species with
diverse biological activities, the transformation from dihydrox-
ybenzoquinone to 20,30,50-trihydroxy-benzene in p-terphenyls
biosynthesis have not been biochemically characterized.
Previously, a series of p-terphenyl O-b-glucuronides echosides
has been isolated from Streptomyces sp. LZ35. Among them, the
major component echoside C (1) showed inhibitory activity against
phenyls are derived from either L-phenylalanine or L-tyrosine, and
the precise mechanism for the biosynthesis of the terphenylquinone
topoisomerase IIa in vitro [6]. For the biosynthesis of echosides in
skeletons, polyporic acid and atromentin, have been clarified. The
strain LZ35, we have recently identified the echosides biosynthetic
gene cluster (ech, GenBank accession number KJ156360) and
characterized EchA as a polyporic acid synthetase [5]. The ech
cluster contains a three-gene operon, including echA, echB and echC,
which are clearly related to BPSS0130, BPSS0131 (encoding a
dehydratase) and BPSS0132 (encoding an isomerase) with amino
acid identities of 61.7%, 55.8% and 48.2%, respectively. Since Brady
and coworkers have reported that heterologous expression of
BPSS0130-132 and BPSS0133 (encoding a methyltransferase) pro-
duced compounds with the identical core structure of echosides
amino acids were deaminated into their corresponding
a-keto acids,
phenylpyruvic acid and 4-hydroxyphenylpyruvic acids, respectively.
Subsequent condensation of two molecules of a-keto acids into the
* Corresponding author.
** Corresponding author. State Key Laboratory of Microbial Technology, Shandong
University, Qingdao, 266237, PR China.
E-mail addresses: wanghaoxin@sdu.edu.cn (H. Wang), yshen@sdu.edu.cn
(Y. Shen).
https://doi.org/10.1016/j.bbrc.2021.04.087
0006-291X/© 2021 Elsevier Inc. All rights reserved.

J. Zhu, M. Liu, J. Deng et al.
Biochemical and Biophysical Research Communications 559 (2021) 62e69
Fig. 1. Chemical structure of echoside C (1) and related 20,30,50-trihydroxy-p-terphenyls from other sources.
[7], we proposed that EchB (GenBank accession number
AHN91925.1) and EchC (GenBank accession number AHN91926.1)
were sufficient to catalyze the conversion of dihydrox-
ybenzoquinone to 20,30,50-trihydroxy-benzene in the biosynthesis
of echosides. Herein, we report the biochemical characterization of
EchB and EchC in the formation of 20,30,50-trihydroxy-benzene core
in echosides biosynthesis, through gene deletion, isolation of in-
termediates and in vitro biochemical experiments. These tailoring
steps were not previously known in p-terphenyls biosynthesis,
which can be used as a probe to identified novel 20,30,50-trihydroxy-
p-terphenyls in future.
was then verified by PCR amplification (Table S2, Fig. S1). To
construct the complementary plasmids, the resultant PCR products
of the echB and echC genes were digested and ligated to the NdeI
and EcoRI sites of pJTU824R, respectively. After verification by DNA
sequencing, the complementary plasmids were introduced into the
mutants SR107DechB and SR107DechC by conjugation. The gener-
ated complementation mutants were verified by PCR using the
primers listed in Table S2.
2.3. Production and analysis of the metabolites in mutants
For secondary metabolites production, SR107 and its mutants
were inoculated on M4 medium in petri dishes and cultivated for 11
days at 30 ꢀC. The culture was diced and extracted with ethyl ac-
etate/methanol/acetic acid 80: 15: 5 (v/v/v) at room temperature.
The crude extract was decanted and concentrated under reduced
pressure, and the extract was dissolved in 1.5 mL methanol. An
2. Materials and methods
2.1. Bacterial strains, plasmids, oligos and culture conditions
Bacterial strains, plasmids, and oligos used in this study are
listed in Tables S1 and S2 in the Supporting Information. The media
and culture conditions for Streptomyces and E. coli strains were the
same as previously reported [5].
aliquot of each extract (10
mL) was analyzed by HPLC-ESI-MS, a
Thermo LTQ velos pro Orbitrap ETD spectrometer equipped with an
HPLC system (SHIMADZU, prominence LC-20AB) using an electro-
spray (ESI) ionization source in negative mode. Full scan mass
spectra were acquired within the range of m/z 150e500 at 30,000
resolution (at m/z ¼ 400) in the Orbitrap. A YMC-pack pro-C18
2.2. Generation of gene deletion and complementation mutants in
SR107
reverse phase column (4.6 ꢁ 250 mm, 5
mm, YMC) was used to
separate samples. Mobile phase A contained 0.1% formic acid (FA) in
water and phase B contained 0.1% FA in acetonitrile. The samples
were eluted at a flow rate of 1 mL/min using a linear gradient as
follows: 0 min, 20% B, 5 min, 35% B, 19 min, 55% B, 20 min, 60% B,
23 min, 100% B, 27 min, 100% B. Samples were monitored at UV
270 nm.
The fosmid containing the entire ech gene cluster was used for
generation of the gene deletion constructs using the l-RED-medi-
ated PCR-targeting method [5]. Briefly, an apramycin resistant
cassette was amplified from pIJ773 using the primers (DP3BPTF/
DP3CPTR, DP3BPTF/DP3BPTR, DP3CPTF/DP3CPTR, Table S2). These
fragments were individually transformed into E. coli BW25113/
pKD46/fosmid, respectively. The resulting constructs were indi-
vidually electroporated into E. coli ET12567/pUZ8002, which were
conjugated with SR107 to produce the deletion strains
2.4. Fermentation and isolation of compounds 4 and 5
SR107
D
echBC, SR107
D
echB, and SR107
D
echC. Each of the mutants
The fermentation of SR107DechB was performed on M4 medium
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J. Zhu, M. Liu, J. Deng et al.
Biochemical and Biophysical Research Communications 559 (2021) 62e69
at 30 ꢀC for 11 days. The 6-Liter fermented agar cakes were diced
and extracted three times overnight with EtOAc/MeOH/AcOH
(80:15:5, v/v/v) to afford a crude extract. The extract was subjected
to Sephadex LH-20 (100 g) eluted with CH2Cl2eMeOH (1:2, v/v) to
afford 10 fractions: Fr.1e10. Fr.9 (35 mg) was subjected to MPLC
(30 g, RP-18 silica gel, 15%, 30%, 40%, 60% CH3CN and 100% MeOH,
100 mL each) to afford five fractions: Fr.9a - 9e. Fr.9a (22 mg) was
purified by semi-preparative HPLC (Agilent, ZORBAX Eclipse XDB-
C18, 9.4 ꢁ 250 nm, UV 274 nm) eluted with 37% CH3CN to afford
2.8. In vitro assay for the activity of EchB and EchC
In the assays of the EchB activity, the reaction contained 50 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 300 mM NaCl, 2 mM NADPH (or
NADH), 6
(5). After incubation at 30 ꢀC for overnight, 100
added to the samples (50 l) and then centrifuged at 16,000 g to
mM EchB, 100
mM polyporic acid (2) or 100
mM echoside G
l methanol was
m
m
pellet the protein. The supernatant of each sample was analyzed by
LC-MS as described for the analysis of the metabolites in mutant
strains. In the assays of the coupled EchB and EchC activity, the
reaction contained 50 mM Tris-HCl (pH 7.0), 10 mM MgCl2, 300 mM
4 (tR ¼ 5.0 min, 2 mg). The fermentation of SR107
formed on M4 medium (10 L) at 30 ꢀC for 11 days. The culture was
extracted as mentioned for SR107 echB to give the crude extract,
DechC was per-
D
NaCl, 2 mM NADPH (or NADH), 10 mM EchB, 10 mM EchC (EchC-
which was then partitioned between H2O and EtOAc. The H2O
extract was further partitioned between H2O and EtOAc/MeOH
(EM, 5:1) to afford EM extract. The EM extract (3.54 g) was sub-
jected to MPLC (140 g, RP-18 silica gel, 30%, 35%, 40%, 45%, 50%
CH3CN, and 100% MeOH, 1 L each) to afford eight fractions: Fr.1e8.
Fr.2 (236 mg) was subjected to Sephadex LH-20 (140 g) eluted with
MeOH, and followed by MPLC (30 g, RP-18 silica gel, 25%, 28%, 30%,
35% CH3CN and 100% MeOH, 100 mL), and further purified by semi-
preparative HPLC eluted with 45% CH3CN to afford 5 (tR ¼ 10.3 min,
12.8 mg).
H40A or EchC-R93A). After incubation at 30 ꢀC for certain time,
each of the samples was prepared as mentioned above, and
analyzed by LC-MS using a linear gradient of 5%e100% acetonitrile
(0.1% FA) over 10 min and 100% acetonitrile (0.1% FA) for 5 min.
3. Results and discussion
3.1. EchB and EchC involved in the formation of 20,30,50-trihydroxy-
benzene core
In order to confirm the function of EchB and EchC in formation
of 20,30,50-trihydroxy-benzene core, we replaced echBC, echB and
echC by an apramycin resistance cassette in strain SR107 to
2.5. Acid hydrolysis of compound 1
A solution of 10% HCl (1 mL), tetrahydrofuran (1 mL), and
compound 1 (8.3 mg) was stirred for 45 min at 80 ꢀC. The reaction
was quenched by addition of 100 mL H2O and extracted with EtOAc.
The solvent was evaporated, and the residue (3 mg) was chroma-
tographed on HPLC (Agilent, ZORBAX Eclipse XDB-C18,
9.4 ꢁ 250 nm, UV 274 nm) eluted with 50% CH3CN to afford 7
(tR ¼ 7.0 min, 1 mg).
generate mutants SR107DechBC, SR107DechB and SR107DechC,
respectively (Fig. S1). Strain SR107 was derived from Streptomyces.
sp. LZ35 through deletion of the four native PKS gene clusters,
which was expected to facilitate the detection of biosynthetic in-
termediates [8]. The metabolites of SR107 and the mutants were
analyzed by LC-HRMS (Fig. 2). All mutants completely abolished the
production of echoside C (1), the major component of echosides.
Furthermore, complementation of echB and echC in the corre-
sponding mutants restored echoside C production (Fig. S3), sug-
gesting the involvement of echB and echC in echosides biosynthesis.
2.6. Expression and purification of EchB and EchC
The amplified echB gene was digested with NdeI and XhoI, and
cloned into pET22b. The echC gene was cloned into pET22b at the
NdeI and XhoI sites using sequence- and ligation-independent
cloning (SLIC) method as previously described for echA [5]. The
single colonies of E. coli BL21 (DE3) containing pET22b-echB or
pET22b-echC were inoculated in LB medium containing ampicillin
SR107DechBC produced two extra peaks that were absent in the
control strain SR107, and an increased peak at a retention time of
24 min which was identical to the standard polyporic acid (2)
(Fig. 2) [5]. Since polyporic acid was expected to be the only in-
termediate accumulated in SR107DechBC, we first focused our
attention on the two extra peaks. However, only one extra peak (4)
was identified and obtained in large scale fermentation of
(100
density (OD600) reach 0.6e0.8. After addition of 0.1 mM IPTG (iso-
propyl- -thiogalactopyranoside), the culture was further incu-
m
g/mL), and the culture was incubated at 37 ꢀC until the cell
SR107DechBC. HRMS spectrum showed an m/z of 309.0763 for 4
b
-D
[M(C18H14O5) e H]- (Fig. S13). The structure of 4 was elucidated by
extensive one- and two-dimensional (2D) NMR spectroscopy
(Fig. 2, Table S3, Figs. S9eS13). The two monosubstituted phenyl
bated at 16 ꢀC for 20 h. The induced cells were harvested by
centrifugation (4000 g, 30 min at 4 ꢀC) and resuspended in lysis
buffer (25 mM Tris-HCl, 300 mM NaCl, pH 8.0), and the suspension
was sonicated on ice. The soluble fraction was collected by centri-
fugation (13,000 g, 30 min at 4 ꢀC) and applied onto a Ni-NTA
column (GE Healthcare, Ni Sepharose™ 6 Fast Flow). The column
was washed with 15 mM imidazole and eluted with 200 mM
imidazole in lysis buffer. The concentrated protein was exchanged
by the lysis buffer using an Amicon ultra centrifugal filter (10000
MWCO for EchB, 3000 MWCO for EchC and its mutants, EMD
Millipore).
rings of 4 were identified on the basis of aromatic protons at d 7.24
(t, J ¼ 7.3 Hz, 1H), 7.35 (t, J ¼ 7.4 Hz, 2H), 7.42 (t, J ¼ 7.4 Hz, 2H), 7.53
(t, J ¼ 7.3 Hz, 1H), 7.84 (d, J ¼ 7.5 Hz, 2H), and 8.04 (d, J ¼ 7.5 Hz, 2H).
HMBC correlations from H-2 and H-6 to C50, form H-200 and H-6” to
C10, and from H-40 to C10, C30, C50 and C6’ established the structure
of 4 (Fig. 2), which was proposed to be derived from polyporic acid
by unknown metabolism. In addition, SR107DechB produced the
same intermediates as SR107 echBC, indicating that the reaction
D
catalyzed by EchB occurs before that by EchC, which means that
polyporic acid (2) is probably the substrate of EchB.
2.7. Site-directed mutagenesis
SR107DechC produced one extra peak (5) along with the accu-
mulated polyporic acid (2) (Fig. 2). 5 was isolated from a large scale
The predicted substrate binding site His40 of EchC was mutated
to Ala40 or Asn40, and Arg93 of EchC were changed to Ala93, Glu93, or
Gln93 by PCR using the QuikChange Site-Directed Mutagenesis Kit
(Stratagene). The primers used in the experiments are listed in
Table S2. All mutated sites were confirmed by DNA sequencing.
Purification of the mutated EchC proteins were performed as
described for EchC wild type.
(10 L) cultivated SR107DechC. HRMS analysis of 5 gave an m/z
295.0969 for [M ꢂ H]- (Fig. S19), with a molecular formula of
C
18H16O4 (calculated, 295.0970). The 1H NMR spectrum revealed
that 5 had two monosubstituted phenyl units on the basis of aro-
matic protons at
d
7.24 (m, 4H), 7.33 (t, J ¼ 7.6 Hz, 4H), and 7.41 (d,
J ¼ 7.4 Hz, 2H), which was supported by the 13C NMR spectrum and
HSQC correlations (Table S4). HMBC correlations from H-10 to C100,
64

J. Zhu, M. Liu, J. Deng et al.
Biochemical and Biophysical Research Communications 559 (2021) 62e69
Fig. 2. (A) LC-HRMS analysis of the metabolites of SR107 and various mutant strains (HRMS spectra were provided in Fig. S2). (B) Chemical structure of echosides F (4) and G (5).
C200, C600, and C20, from H-20 to C10, C60, and C100, from H-2 and H-6 to
C40 allowed the identification of the central ring, even though two
carbon signals were absent in the NMR spectra (Figs. S15eS18). It
can be noticed that compound 5 has three chiral centers, indicating
stereospecificity in the formation of 5. The X-ray crystallography
fully confirmed the proposed structure of 5 and provided the ab-
solute configuration as shown (Fig. S4, deposition number CCDC
1878312). Based on the structure of the biosynthetic intermediates
identified, a possible route to 20,30,50-trihydroxy-p-terphenyl (3)
was proposed. Polyporic acid (2) was stereospecifically reduced by
EchB with addition of four hydrogen atoms, to generate 5.
Following a dehydration reaction catalyzed by EchC, 5 was trans-
formed to 3.
same “Rossmann fold” motif for nucleotide binding, though the
members exhibit low sequence similarities (20%e30%) [9,10]. Based
on differences in the highly conserved glycine-rich nucleotide
binding motif and the catalytic tetrad, SDRs have been classified
into six families by the SDR nomenclature initiative: Classical,
Extended, Intermediate, Divergent, Complex, Atypical SDRs, and
Unassigned, using Hidden Markov Models (HMMs) [9]. EchB was
assigned in the “Unassigned” family by sequence search on the SDR
web page http://sdr-enzymes.org/ [9]. A multiple sequence align-
ment of putative EchB proteins from various organisms was per-
formed using ClustalO. The alignment revealed the presence of the
conserved nucleotide binding motif (TGxxGxxA) in the N-terminal
region, which is different from other classified SDR members, and
the common catalytic triad YxxxK (Fig. S5).
To investigate the function of EchB, recombinant EchB with C-
terminal His6-tag was purified from E. coli BL21 (DE3) to homoge-
neity (Fig. S6A). Purified EchB was able to convert 2 to 5, in the
presence of NADPH or NADH, with the negative control using
boiled EchB as the catalyst (Fig. 3). This revealed that EchB has dual
3.2. Characterization of EchB
Analysis of the EchB protein sequence showed that it belongs to
the short chain dehydrogenase/reductase (SDR) superfamily. This
family is one of the largest superfamilies, known for having the
65

J. Zhu, M. Liu, J. Deng et al.
Biochemical and Biophysical Research Communications 559 (2021) 62e69
coenzyme specificity, with a slight preference for NADPH. The
EchB-catalyzed reverse reaction was examined with 5 as the sub-
strate under the condition where NAD(P)H was replaced with
NAD(P)þ, showing a visible production of 2 (Fig. 3). Consequently,
these results established that EchB could catalyze reduction of 2
into 5 as expected. It was worth noticing that the reduction of 2
catalyzed by EchB consisted two steps of reduction, requiring two
NAD(P)H molecules. Moreover, the reactions were not only ste-
reospecific but also region specific.
central hole as shown in the homology modeling structure of EchC
(Fig. S8). In order to clarify the catalytic role of EchC, we constructed
five mutants of EchC including EchC-H40A, EchC-H40N, EchC-
R93A, EchC-R93E, and EchC-R93Q, then purified two high soluble
proteins, EchC-H40N and EchC-R93A (Fig. S6C). Both of the product
profiles of the EchC-H40N and EchC-R93A reactions were the same
as the reaction with EchB only, suggesting His40 and Arg93 were
important to the dehydration activity of EchC (Fig. 4B).
3.4. Pathway for the formation of 20,30,50-trihydroxy-p-terphenyl
3.3. Coupled reaction catalyzed by EchB and EchC
Based on the results, we proposed a new version of pathway for
the formation of 20,30,50-trihydroxy-p-terphenyl (3) in echosides
biosynthesis (Fig. 4D). This transformation has gone through
reduction and dehydration, which include three tailoring steps
acting on the dihydroxybenzoquinone core: polyporic acid (2)
synthesized by EchA is reduced by EchB to generate intermediate 6;
EchC performs a dehydration using 6 as substrate to give 7; and 7
undergoes another reduction to afford 3 catalyzed by EchB. Absent
of EchC, further reduction of 6 mediated by EchB would resulted in
a dead-end shunt product 5, which cannot be dehydrated by EchC.
Previously, The transformation from 1,3,6,8-tetrahydroxy
naphthalene to 1,8-dihydroxynaphthalene (DHN) includes a suc-
cession of two reduction and two dehydration steps, involving two
different SDR enzymes (tetrahydroxynaphthalene reductase 4HNR
and 3HNR), and one NTF2-like enzyme scytalone dehydratase [12].
A single 3HNR (MgTHNB) catalyzed both reduction steps has also
been reported [15]. Another DHN melanin-like pathway has also
been identified in anthrahydroquinones biosynthesis [16].
Sequence analysis revealed that EchC belongs to the nuclear
transport factor 2 (NTF2)-like superfamily. Members of this family
share a common fold but with no conserved sequence motif. The
enzymatically active NTF2-like proteins include scytalone dehy-
dratases, delta-5-3-ketosteroid isomerases, polyketide cyclase
SnoaL, limonene-1,2-epoxide hydrolase, and so on [11]. However,
contrary to our proposal, in vitro experiment using purified EchC
and 5 showed no bioconversion result, indicating 5 is probably not
an intermediate on the paths to 3.
By analogy to 1,3,6,8-tetrahydroxynaphthalene formation in
melanin biosynthesis [12], the formation of 3 was then proposed to
proceed by a succession sequence of reduction, dehydration, and
another reduction (Fig. 4D). To validate this hypothesis, EchB and
EchC were incubated with 2 and NADPH. Subsequent HPLC-MS
analysis revealed the formation of two products after 30 min of
reaction (Fig. 4A, Fig. S7). The newly formed ones showed molec-
ular masses corresponding to 3 and 7 (Fig. 4C). In addition, the
retention time of 7 was identical to the standard one, which was
obtained by spontaneous oxidation of the product (3) of acid hy-
drolysis of echoside C (1). The combined evidence suggests EchB
and EchC function together to catalyze the transformation of 2 into
3. Interestingly, the product profile of the EchB and EchC coupled
reaction changed along with the incubation time (Fig. 4A).
Extended the reaction time to 1 and 7 h, we found 7 was gradually
consumed, resulting in the accumulation of only 2 ultimately. This
could be explained by the spontaneous oxidation of 3 along with
the exhaustion of NADPH in vitro. It also indicated that EchC
probably catalyzed reversible dehydration reaction.
When we examined the products of EchB reaction for 20 min by
HPLC-MS, we observed a minor peak with an increased mass of
2 Da appeared, which was exactly in agreement with the expected
intermediate 6 (Fig. 4B and C). Trace levels of 3 and 7 were also
detected in the products of EchB reaction, either because 6 would
spontaneously dehydrate, or EchB probably also poorly catalyzed
the dehydration of 6. Previously, another SDR family enzyme tri-
hydroxynaphthalene reductase (3HNR), which catalyzes the
NADPH dependent reduction of 1,3,8-trihydroxynaphthalene to
vermelone in melanin biosynthesis pathway, has been reported to
catalyze the scytalone dehydration reaction with poor activity [13].
In the presence of EchC, the consumption of 2 in the reaction
significantly increased, without 5 generated (Fig. 4B), indicating the
dehydration of 6 by EchC was faster than reduction of 6 by EchB. As
soon as 6 was generated from 2 by EchB, it was withdrawn by EchC
from the equilibrium by way of dehydration to 7. When EchC was
Although most of the 20,30,50-trihydroxy-p-terphenyls were
found in fungi, and atromentin synthase was also first character-
ized in the fungi homobasidiomycete Tapinella panuoides [17]. The
biosynthesis mechanism of 20,30,50-trihydroxy-p-terphenyls in
fungi have not been reported. In genomic database, the EchA ho-
mologues (tridomain NRPS) were widely found in fungi and bac-
teria, indicating the quinones backbone were transformed by the
same mechanism. However, we only found coupled of SDR and
NTF2-like tailoring genes in bacteria, mainly in Streptomyces and
Burkholderia. This indicated that either the fungi evolved an alter-
native pathway of this kind of natural products or the biosynthetic
genes located in separated areas of the genome. For the latter
possibility, it is hard to predict the cadidate genes due to the small
size, low conservation, and wide distribution of SDR and NTF2-like
enzymes. There was an SDR gene but no NTF2-like gene nearby the
echA homologue in some strains of Aspergillus taichungensis (the
same genus but not the same strains reported as the 20,30,50-
trihydroxy-p-terphenyls producers). The SDR was annotated as 6-
phosphogluconate dehydrogenase-like protein, showing no simi-
larity with EchB when aligned by BlastP. Whether the SDR gene
related to the production of 20,30,50-trihydroxy-p-terphenyls needs
to be explored.
4. Conclusion
There is a large number of 20,30,50-trihydroxy-p-terphenyls,
which are mainly isolated from fungi, few cases from bacteria and
the moss [3]. So far, only two types of 20,30,50-trihydroxy-p-ter-
phenyls discovered from bacteria have been linked to their
biosynthetic gene clusters [5,7]. In this work, combining the genetic
and biochemical data, we established the pathway from polyporic
acid (2) to 20,30,50-trihydroxy-p-terphenyl (3) in echosides biosyn-
thesis and demonstrated the importance of the reaction sequence
in determining the pathways. Echosides represented the first bio-
chemically characterized 20,30,50-trihydroxy-p-terphenyl cluster
that involves SDR and NTF2-like enzymes in the 20,30,50-trihydroxy-
10-fold less than EchB (1 mMe10 mM), 2 was still fully transformed
to 3,7 (Fig. S7). Due to their instability, we failed to obtain sufficient
amounts of purified 3, 6 and 7 to do further study.
Structural prediction and function annotation of EchC by the I-
TASSER server [14]gave several possible substrate binding residues
and active sites. Excluding the inconsistent active sites predicted
from different templates, we chose two conserved substrate bind-
ing sites candidates (His40 and Arg93) in the top 4 homologues
(Table S5) to construct mutants. The two residues pointed to the
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J. Zhu, M. Liu, J. Deng et al.
Biochemical and Biophysical Research Communications 559 (2021) 62e69
Fig. 3. In vitro activity assays of EchB using compound 2 or 5 as substrate. (A) The reaction catalyzed by EchB. (B) HPLC analysis of EchB reactions.
67

J. Zhu, M. Liu, J. Deng et al.
Biochemical and Biophysical Research Communications 559 (2021) 62e69
Fig. 4. In vitro activity assays of EchB and EchC using compound 2 as substrate. (A) LC-HRMS analysis of reaction products of EchB/EchC coupled assays after different reaction time.
(B) LC-HRMS analysis of reaction products of EchC mutants. (C) HRMS spectra of compounds 3, 6 and 7. (D) The coupled reaction catalyzed by EchB and EchC.
benzene formation.
University, China [2018JC004]; and the Program for Changjiang
Scholars and Innovative Research Team in University, China
[IRT_17R68].
Accession codes
EchB (GenBank accession number AHN91925.1) and EchC
(GenBank accession number AHN91926.1).
Declaration of competing interest
The authors declare no competing financial interest.
Acknowledgements
Funding
This work was supported by the Key Research and Development
Project of Shandong Province, China [2019GSF108212]; the Na-
tional Natural Science Foundation of China, China [31500030]; the
Shandong Province Natural Science Foundation, China
[BS2015SW025]; the China Postdoctoral Science Foundation, China
[2016M602131]; the Fundamental Research Fund of Shandong
We thank Zhifeng Li, Haiyan Yu and Jingyao Qu from State Key
laboratory of Microbial Technology of Shandong University for help
and guidance in liquid chromatography-tandem mass spectrom-
etry. We thank Amanda Miller for grammar check on the
manuscript.
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J. Zhu, M. Liu, J. Deng et al.
Biochemical and Biophysical Research Communications 559 (2021) 62e69
185e188.
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[10] K.L. Kavanagh, H. Jornvall, B. Persson, et al., Medium- and short-chain dehy-
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2021.04.087.
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