New insights into the disulfide bond formation enzymes in epidithiodiketopiperazine alkaloids

Article abstract of DOI:10.1039/d0sc06647h

Epidithiodiketopiperazines (ETPs) are a group of bioactive fungal natural products and structurally feature unique transannular disulfide bridges between α, α or α, β carbons. However, no enzyme has yet been demonstrated to catalyse α, β-disulfide bond formation in these molecules. Through genome mining and gene deletion approaches inTrichoderma hypoxylon, we identified a putative biosynthetic gene cluster of pretrichodermamide A (1), which requires a FAD-dependent oxidoreductase, TdaR, for the irregular α, β-disulfide formation in1biosynthesis.In vitroassays of TdaR, together with AclT involved in aspirochlorine and GliT involved in gliotoxin biosynthesis, proved that all three enzymes catalyse not only the conversion of red-pretrichodermamide A (4) to α, β-disulfide-containing1but also that of red-gliotoxin (5) to α, α-disulfide-containing gliotoxin (6). These results provide new insights into the thiol-disulfide oxidases responsible for the disulfide bond formation in natural products with significant substrate and catalytic promiscuities.

Full text of DOI:10.1039/d0sc06647h

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New insights into the disulfide bond formation
enzymes in epidithiodiketopiperazine alkaloids†
Cite this: Chem. Sci., 2021, 12, 4132
d
Huan Liu,‡a Jie Fan, ‡a Peng Zhang,a Youcai Hu,
Xingzhong Liu,a
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
b
ac
*
*
Shu-Ming Li
and Wen-Bing Yin
Epidithiodiketopiperazines (ETPs) are a group of bioactive fungal natural products and structurally feature
unique transannular disulfide bridges between a, a or a, b carbons. However, no enzyme has yet been
demonstrated to catalyse a, b-disulfide bond formation in these molecules. Through genome mining
and gene deletion approaches in Trichoderma hypoxylon, we identified a putative biosynthetic gene
cluster of pretrichodermamide A (1), which requires a FAD-dependent oxidoreductase, TdaR, for the
irregular a, b-disulfide formation in 1 biosynthesis. In vitro assays of TdaR, together with AclT involved in
aspirochlorine and GliT involved in gliotoxin biosynthesis, proved that all three enzymes catalyse not only
the conversion of red-pretrichodermamide A (4) to a, b-disulfide-containing 1 but also that of red-
gliotoxin (5) to a, a-disulfide-containing gliotoxin (6). These results provide new insights into the thiol-
disulfide oxidases responsible for the disulfide bond formation in natural products with significant
substrate and catalytic promiscuities.
Received 5th December 2020
Accepted 28th January 2021
DOI: 10.1039/d0sc06647h
rsc.li/chemical-science
gliotoxin,6–9 sirodesmin PL,10–14 acetylaranotin,15 dithiosilva-
tin,16 sporidesmin7,17 and epicorazine18 bear a disulde bridge
Introduction
across the a and a positions of the DKP skeleton. In contrast,
pretrichodermamide A (1),19 gliovirin,20,21 adametizine A,22,23
aspirochlorine as well as its biosynthetic intermediate24,25 and
lasiodipline D26 carry an irregular disulde bridge across
Natural products containing sulfur display remarkable struc-
tural diversity and have a wide variety of biological activities.1,2
A
special
subgroup
found
in
fungi
includes
epi-
dithiodiketopiperazines (ETPs) featuring
a
cyclodipeptide
(CDP) scaffold, which is decorated by a transannular disulde
bridge. They exhibit a broad range of bioactivities including
anticancer, antibacterial, antifungal, antiviral, immunosup-
pressive and cytotoxic activities.1,3,4 Typically, structural diver-
sity of ETPs possessing different core CDPs is generated by
nonribosomal peptide synthetases (NRPSs) and multiple post-
assembly modications.5 Commonly derived from phenylala-
nine-, tyrosine- and tryptophan-containing CDPs,1 two subtypes
of bioactive ETPs carrying disulde bridges across a, a- or a, b-
positions of the diketopiperazine (DKP) skeleton have been
identied in diverse fungal species (Fig. 1). Representatively,
aState Key Laboratory of Mycology, CAS Key Laboratory of Microbial Physiological and
Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing
100101, P. R. China. E-mail: yinwb@im.ac.cn
b
¨
Institut fur Pharmazeutische Biologie und Biotechnologie, Fachbereich Pharmazie,
¨
Philipps-Universitat Marburg, Robert-Koch-Straße 4, Marburg 35037, Germany.
E-mail: shuming.li@staff.uni-marburg.de
cSavaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, P.
R. China
dState Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union
Medical College, Beijing 100050, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0sc06647h
Fig. 1 Representative fungal ETPs with an a, a- (A) or a, b- (B) disulfide
‡ These authors contributed equally to this work.
bond.
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corresponding a and b positions (Fig. 1). To date, these unique and 1,2-oxazadecaline moiety of 1 as well as its derivatives have
structures have captured the attention of scientists for their shown to be essential for the observed biological activities.21,22
total synthesis27,28 and biosynthesis.1,9,12
biosynthetic route was proposed for gliovirin, a derivative of 1,
A
Previous studies have elucidated many aspects of the in Trichoderma virens, based on genome mining and core gene
molecular mechanism involved in the biosynthesis of gliotoxin deletion experiments.21 It was speculated that the FAD-
from Aspergillus fumigatus, including cyclo-L-Phe-L-Ser genera- dependent oxidoreductase Glv4 or Glv16 was possibly involved
tion by an NRPS enzyme GliP, hydroxylation by cytochrome in the a, b-disulde bridge formation. However, data for neither
P450, sulfur moiety incorporation by the addition of glutathione its detailed biosynthesis nor for the a, b-disulde formation was
(GSH) and its sequential degradation to the dithiol reported. In this study, we identied 1 from a fungicolous
precursor.5,29,30 A avin adenine dinucleotide (FAD)-dependent fungus Trichoderma hypoxylon and proposed
a possible
oxidoreductase GliT is responsible for the oxidation of the biosynthetic pathway by genome mining and deletion of two
dithiol group yielding the a, a disulde-containing gliotoxin.31,32 targeted genes. Biochemical investigations proved that a FAD-
In addition, biosynthesis of several ETPs including sirodesmin dependent oxidoreductase, TdaR, was responsible for the a, b-
PL from Leptosphaeria maculans and acetylaranotin from disulde formation in the biosynthesis of 1. Further in vitro
Aspergillus terreus was extensively studied by multiple genetic assays demonstrated that three oxidases TdaR, AclT and GliT
and biochemical approaches.12–15 The halogenated aspiro- from different pathways catalysed both a, a- and a, b-disulde
chlorine was also investigated with respect to its crucial roles in bond formation in the biosynthesis of fungal ETP alkaloids.
the formation of a cyclo-L-Phe-Gly containing ETP from cyclo-L-
Phe-L-Phe.24 Although the interesting a, b-disulde bridge
formation was speculated to be catalysed by a GliT homolog,
Results
Identication of the biosynthetic gene cluster for
pretrichodermamide A
AclT, no experimental evidence was provided.
To identify the enzyme involved in a, b-disulde bridge
formation, we focused on 1 reported in Trichoderma sp.
BCC5926, which exhibits antibacterial activity towards Myco-
bacterium tuberculosis H37Ra.19 The typical a, b-disulde bridge
To identify the ETP 1, we examined the secondary metabolites
of previously reported fungus T. hypoxylon CGMCC 3.17906,
because several ETP compounds including trichodermamide A
Fig. 2 Pretrichodermamide A (1) biosynthetic gene cluster (A), LC-MS analysis of T. hypoxylon strains (B) and the proposed biosynthetic pathway
for 1 (C).
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(2), aspergillazine A and aspergillazine C have been identied by
our group.33 Spontaneous conversion of 1 to 2 was demon-
strated by Seephonkai et al. due to the instability of 1 under
alkaline conditions or even in methanol.19 Therefore, we believe
that 1 should exist in this strain under controlled culture and
extraction conditions. Herein, T. hypoxylon was cultivated in
rice medium and the ethyl acetate extracts were analysed on
HPLC (Fig. S1 in the ESI†). As expected, one predominant peak
with an [M + H]+ ion at m/z 499.0845 was detected in a 14 day-old
rice culture, which was subsequently identied as 1 aer
isolation and structure elucidation by comparing to reported
data (Fig. S2, S3 and Table S3†).19 The spontaneous conversion
of 1 to 2 was observed, even under cultivation conditions and
during isolation (data not shown). This result indicated that 1
was the natural ETP metabolite in T. hypoxylon.
Fig.
3
Sequence clustering and phylogenetic analysis of FAD-
dependent oxidoreductases involved in thiol-disulfide formation. Four
distinct clades are shown in different colors with their representative
sulfur-containing products. Romidepsin and holomycin were formed
by DepH (clade I) and HlmI (clade II), respectively. TrxR (clade III) is
a thioredoxin reductase responsible for the reduction of disulfide.
TdaR, together with GliT (involved in gliotoxin biosynthesis) and AclT
(involved in aspirochlorine biosynthesis), was located in clade IV with
other potential fungal thiol-disulfide oxidases. Amino acid sequences
were obtained from the GenBank database.
To determine the biosynthetic pathway of 1, the genome of T.
hypoxylon was sequenced and the dra genome sequence was
used for the prediction of putative gene clusters by using Anti-
SMASH.34 Based on its ETP structural character, 1 should be
derived from cyclo-L-Phe-L-Phe (3), which is expected to be
assembled by an NRPS.35 One of the eleven NRPS genes
T_hypo_11188, termed tdaA, within a large 49.6 kbp cluster
(Fig. 2A and Table S4†), contains a T–C–A–T–C domain struc-
ture, being similar to the core enzymes AtaP15 and AclP24 in
phenylalanine-containing ETP biosynthesis (abbreviations for
NRPS domains are as given before35). TdaA shares a sequence
similarity of 31% with GliP9 from A. fumigatus and 88% with
Glv21 21 from T. virens on the amino acid level (Table S4†). To
prove its function, tdaA was deleted from the genome by PEG-
mediated protoplast transformation using hygromycin B as
a selection marker.36 LC-MS analysis revealed complete disap-
pearance of ETP products 1 and 2, as well as the NRPS product
3, indicating that TdaA acts as the core enzyme for the biosyn-
thesis of 1 and 2 (Fig. 2B). To nd the candidate gene for the a,
b-disulde bridge formation in 1, further blast analysis was
performed using the disulde bond formation enzyme GliT in
the gliotoxin biosynthetic pathway as a probe.32 Two putative
FAD-dependent oxidoreductases TdaE (T_hypo_11193) and
TdaR (T_hypo_11206) are located in the pretrichodermamide A
(tda) cluster. Alignments with known oxidases for thiol-
disulde formation showed the existence of a conserved active
binding site CLFC (CXXC) box in TdaR but not in TdaE
(Fig. S4†). Therefore, we performed tdaR deletion according to
the aforementioned method for tdaA, leading to complete
disappearance of 1 and 2, but accumulation of 3 as indicated by
the analysis of extracted ion chromatograms (EICs) (Fig. 2B).
This proved the involvement of TdaR in the conversion of 3 to 1
and 2. Inspection of the EICs of the culture extracts and struc-
ture analysis implied more conversion steps between 3 and 1.
Thus, a biosynthetic pathway of 1 and 2 was proposed as shown
in Fig. 2C. Briey, the DKP core is assembled by NRPS TdaA (i),
followed by hydroxylations at a and b positions by a putative
cytochrome P450 TdaS (ii). Aer sulfur incorporation and
sequential degradation (iii), TdaR catalyses the a, b-disulde
bond formation (iv). Subsequently, further hydroxylations cat-
alysed by multiple cytochrome P450s and methylations cata-
formation of 1 with a 1,2-oxazadecaline moiety and methoxy
groups (v). Lastly, spontaneous degradation results in the
transformation of 1 to 2 (vi).
TdaR responsible for a, b-disulde bond formation
To address whether TdaR is involved in the a, b-disulde bond
formation, we performed in vitro biochemical characterisation.
Previous studies reported that disulde bond formation was
catalysed by FAD-dependent oxidoreductases from different
bacterial and fungal sources.37 Hence, a sequence similarity
network of TdaR homologs including 370 FAD-dependent
oxidoreductases was generated which led to the identication
Fig. 4 Analysis of TdaR, AclT and GliT on SDS-PAGE (A) and enzyme-
lysed by different methyltransferases (MeTs) lead to the mediated disulfide bond formation in the biosynthesis of 1 and 6 (B).
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of four distinct clades at an e-value threshold of 80 (Fig. 3 and
S5†).38 TdaR belongs to clade IV which includes GliT and 81
other not yet fully identied fungal enzymes possibly from ETP
biosynthetic pathways. Prototypes of the other three clades are
DepH (clade I),39 HlmI (clade II)40 and TrxR (clade III)41 from
Chromobacterium violaceum, Streptomyces clavuligerus and
Escherichia coli, respectively. Interestingly, proteins from clade
I, II and IV presumably oxidize small molecules yielding disul-
de bonds, while clade III probably acts as reductases for
intracellular redox regulation.
To investigate its function, the 933 bp long coding sequence
of tdaR comprising three exons was cloned from cDNA of T.
hypoxylon and overexpressed in E. coli BL21(DE3) cells. The
recombinant His6-tagged protein was puried with the aid of
Ni-NTA agarose resin to near homogeneity which was conrmed
on SDS-PAGE (Fig. 4A), yielding 5.1 mg of puried TdaR per liter
of bacterial culture. Due to the instability of the unidentied
substrate of TdaR for the a, b-disulde bond formation, we
proposed that this enzyme could also catalyse the conversion of
reduced pretrichodermamide A (red-pretrichodermamide A, 4)
to 1. Therefore, 1 was chemically reduced to 4 using dithio-
threitol (DTT) to provide an appropriate substrate for the
enzyme assays (Fig. S6†). HPLC analysis revealed a complete
ꢀ
conversion of 0.5 mM 1 to 4 by 1 mM DTT at 30 C for 30 min
(Fig. 5A and S6†). Subsequently, this mixture was directly
incubated with 0.1 mM puried TdaR at 37 ꢀC for 30 min. As
expected, the enzyme assay led to the formation of 1 which was
veried by comparing the retention time with that of an
authentic standard (Fig. 5A(i, ii and viiii)). This proved that the
FAD-dependent oxidoreductase TdaR was responsible for the a,
b-disulde bond formation in the biosynthesis of 1. Further-
more, the time course of this reaction was examined and
monitored by HPLC, indicating that 17% of 0.5 mM 4 was
consumed by 0.1 mM TdaR in 5 min (Fig. S7†). However, no
conversion of 4 could be observed with heat-denatured TdaR or
without O2 in the glove box (Fig. 5A(i and iii)). This indicated
a mechanism of a, b-disulde bond formation similar to GliT-
catalysed a, a-disulde bond formation, which required O2.
Accordingly, we proposed that initial transformation occurred
in TdaR from the oxidized form (disulde) to reduced form
(dithiol) by the putative CLFC redox system. Subsequently,
electron pairs were transferred to FADox from the active site of
TdaR. FADred is then reoxidized by O2 to form H2O2, leading to
the concomitant production of the disulde-containing product
(Fig. 4B and 6).
Fig. 5 HPLC analysis of enzyme assays of TdaR, AclT and GliT with 4
(A) or 5 (B). UV absorption at 254 nm was illustrated.
Fungal thiol-disulde oxidases catalysed both a, a- and a, b-
disulde bond formation
In addition to TdaR, AclT was also predicted to catalyse an a, b-
disulde bond formation in the biosynthesis of aspirochlorine
in Aspergillus oryzae.24 However, no detailed investigation on
AclT has been reported until now. We presumed that AclT could
also catalyse the a, b-disulde bond formation in the biosyn-
thesis of 1. Therefore, the aclT ORF (945 bp) was amplied from
A. oryzae cDNA and cloned into an E. coli expression vector
pET28a(+)SUMO. The resulted plasmid pYHL87 was introduced
into BL21(DE3) cells for protein overproduction, leading to
11.0 mg of puried AclT per liter of bacterial culture. It showed
a yellow color and was conrmed on SDS-PAGE with the pre-
dicted size of 50.4 kDa (Fig. 4A). Subsequently, incubation of 4
To directly conrm the oxidation of TdaR on 4, 4 was puried
from the mixture of 1 with tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) as
a reducing agent on a semi-
preparative HPLC (acetonitrile/H2O, 35 : 65) (Fig. S9†). Incuba-
tion of TdaR with the puried 4 revealed a complete conversion
of 4 to 1 aer incubation at 37 ꢀC for 30 min (Fig. S10A†).
However, the formation of 1 was also observed in the negative
control containing heat-denatured TdaR and 4, demonstrating
the spontaneous disulde formation. In comparison, disulde
formation in 1 catalysed by TdaR was more efficient than the
spontaneous oxidation.
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from 1 liter of bacterial culture and subsequently incubated
with 4 and 5 in a similar way to TdaR and AclT. In accordance
with the previous study, GliT acts as a FAD-dependent oxidase
for the a, a-disulde bond formation to generate 6 from 5 aer
5 min incubation (Fig. 5B and S8†).32 Unprecedentedly, incu-
bation of 4 with GliT also led to the formation of 1, as demon-
strated by HPLC analysis (Fig. 5A(vi and vii)). This conrmed
that the known a, a-disulde bond related enzyme GliT also
catalysed the a, b-disulde bond formation. The time course of
GliT-catalysed oxidation of 4 was monitored by HPLC, revealing
that an increasing amount of 4 ranging from 28–82% was
consumed by 0.1 mM GliT from 5 min to 30 min (Fig. S7†).
Discussion
As a ubiquitously distributed element, sulfur is essential for
Fig. 6 Proposed mechanism of disulfide bond formation in ETP
alkaloids by FAD-dependent thiol-disulfide oxidases.
A transient mediating amino acid, peptide, and RNA synthesis in primary
metabolites and secondary metabolites.42–44 There are various
examples of natural products containing sulfur atoms as key
units for biological functions. In the past decades, ETPs have
garnered considerable attention regarding their biosynthesis,
chemical synthesis and biological application, because the
disulde group contributes promising activities probably by
promoting redox cycling, formation of reactive oxygen species
and protein conjugation.45,46 Mostly, the sulfur bridge func-
tionality of ETPs is attached to the a, a-positions or a, b-posi-
tions of the DKP core in fungi. Signicant progress in
understanding the biosynthetic pathway of a, a-disulde-
containing 6 has been achieved through genetic and biochem-
ical approaches.5,29,30,32,37 However, fungal a, b-disulde-
containing metabolites have been rarely investigated with
respect to their biosynthetic mechanism. Although gene clus-
ters coding for the biosynthesis of a, b-disulde-containing
aspirochlorine and gliovirin have been predicted, the required
biochemical functions have remained elusive.21,24
mixed-disulfide bond was generated between substrate and enzyme
(A and B) leading to a FAD-4a-thiol adduct (C). Subsequently, the
disulfide in the ETP alkaloid was formed with concomitant reduction of
FAD (D).
with 0.1 mM AclT was carried out in a similar way to TdaR. HPLC
analysis revealed 1 as the enzymatic product, proving AclT-
catalysed a, b-disulde bond formation (Fig. 5A(iv and v)).
Further time course of AclT-catalysed reaction revealed that
increasing amounts (18–72%) of compound 4 were consumed
by 0.1 mM AclT from 5 min to 30 min (Fig. S7†). In addition,
incubation of heat-denatured AclT with the puried 4 also
revealed that a trace amount of 1 is formed spontaneously
(Fig. S10A†). This was consistent with the enzyme assay of TdaR
with 4, indicating that AclT and TdaR performed the same
function in the a, b-disulde bond formation.
Notably, bioinformatics study showed that TdaR and AclT
involved in the a, b-disulde bond formation were located in
clade IV with several known a, a-disulde bond-forming
enzymes including GliT,9,32 AtaTC15 and SirT12 (Fig. 3 and S5†).
The close phylogenetic relationship triggered our interest to test
if TdaR and AclT could also catalyse a, a-disulde bond
formation. Thus, reduced gliotoxin (red-gliotoxin, 5) was
acquired by co-incubation of gliotoxin (6) with DTT at 30 ꢀC for
30 min or puried from a mixture containing 6 and TCEP
(Fig. S6 and S9†). Interestingly, we observed that 5 (0.5 mM) was
readily converted into 6 in the presence of TdaR (0.1 mM) or AclT
(0.1 mM) aer 5 min (Fig. S8†). 5 was completely transformed to
6 in 30 min by both TdaR and AclT (Fig. 5B, S8 and S10B†).
These results supported our hypothesis that TdaR and AclT
catalyse not only a, b- but also a, a-disulde bond formation in
the biosynthesis of ETP alkaloids. In addition, TdaR and AclT-
In this study, we identied an a, b-disulde-containing
metabolite 1, together with its spontaneous desulfurized
product 2, in T. hypoxylon. Genome mining led to the identi-
cation of a tda cluster encoding analogues of all aforementioned
gliotoxin (gli) cluster enzymes and ve additional cytochrome
P450s as well as three methyltransferases (Fig. 2A and Table
S4†).5 Deletion of the NRPS coding gene tdaA and the FAD-
dependent oxidase gene tdaR proved their involvement in the
biosynthesis of 1 and 2 from the CDP 3 (Fig. 2B and C). Plau-
sibly, dihydroxylation of 3, a, b-disulde formation and subse-
quent hydroxylations as well as methylations will yield the ETP
alkaloid 1. Further biochemical investigation of TdaR provided
evidence for its involvement in the metabolism of 3 to 1 and
proved its crucial role in the formation of the a, b-disulde
bridge (Fig. 4B). Thus, our results rst represent an example for
understanding the biosynthesis of a, b-disulde-containing
ETP alkaloids, especially the a, b-disulde formation. Other
pretrichodermamide A-like compounds derived from cyclo-L-
Phe-L-Phe, including pretrichodermamides,22 gliovirin,21
FA2097,47 aspergillazines,48 peniciadametizines49 and aspiro-
chlorine,24 are very likely also formed in a similar way regarding
the installation of the typical a, b-disulde and a 1,2-
catalysed a, a-disulde bond formation with
substrate was more efficient than the a, b-disulde bond
formation.
In analogy, the coding sequence of GliT, which was proved to
catalyse the a, a-disulde bond formation in the biosynthesis of
6, was cloned into pET28a(+)SUMO and overexpressed in E. coli
BL21(DE3). 11.2 mg puried GliT was obtained in a yellow color
5 as the
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oxazadecaline moiety. Therefore, further attempts to mine a, b- Surprisingly, GliT involved in the a, a-disulde bond formation
disulde related enzymes would provide evidence for our was proved to catalyse a, b-disulde bond formation as well.
hypothesis.
Despite their unrelated fungal sources and different natural
Previous study on aspirochlorine biosynthesis has already metabolite pathways, they share obviously common catalytic
implied a putative a, b-disulde related oxidase AclT.24 Herein, ability to produce a, a- or a, b-disulde-containing products.
we proved that AclT is an analogous oxidase of TdaR catalysing Our results provided representative examples of fungus-
the a, b-disulde formation. Further biochemical investigations originated thiol-disulde oxidases catalysing different types of
on TdaR and AclT revealed, interestingly, their dual function of disulde formation in the biosynthesis of ETP alkaloids. It can
catalysing not only the a, b-disulde formation in 1 but also the be speculated that in the near future more fungus-originated
a, a-disulde formation in 6 (Fig. 4B and 5). These results thiol-disulde oxidases would be discovered which catalyse
indicated that a, b-disulde related enzymes also catalysed a, a- both a, a- and a, b-disulde formation, even exhibiting wider
disulde formation, and vice versa. Indeed, enzyme assays specicities towards more substrates.
conrmed that the a, a-disulde related GliT was also able to
catalyse a, b-disulde formation in 1. It can be deduced that
Conflicts of interest
hydroxylations on the CDP core catalysed by P450 enzymes
determine the positions of the two hydroxyl groups and nally There are no conicts to declare.
the type of the disulde bond (a, a or a, b).
Taken together, TdaR, AclT and GliT are dual oxidases
independently forming the a, a- or a, b-disulde bridge in
Acknowledgements
fungal ETP biosynthesis. It might well explain why most fungal We thank Rixa Kraut (University of Marburg) for performing LC-
related FAD-dependent oxidases were located in the same clade MS, and Zhen Cai as well as Xuebing Li (Institute of Microbi-
IV (Fig. 3), no matter they naturally catalyse a, a- or a, b-disul- ology, CAS) for supplying the conditional oxygen-free environ-
de formation. In the phylogenetic analysis of TdaR homologs, ment. We also thank the Center for Ocean Mega-Science,
the unique clade III, exemplied by TrxR from E. coli, catalysed Chinese Academy of Sciences. This work was supported by the
the reduction of a disulde bond in thioredoxin.41 In contrast to National Key Research and Development Program of China
DepH (clade I) from C. violaceum and HlmI (clade II) from S. (2018YFA0900701), National Natural Science Foundation of
clavuligerus involved in intra- or interchenar disulde bonds, China (21778075), the Senior User Project of RV KEXUE (KEX-
TdaR, AclT and GliT (clade IV) catalysed transannular disulde UE2019GZ05), Key Research Program of Frontier Sciences, CAS
bond formation.39,40 They shared a catalytically active CLFC box (ZDBS-LY-SM016), Construction of the Registry and Database of
for redox activation, but different FAD binding sites, i.e. His139 Bioparts for Synthetic Biology, CAS (ZSYS-016), the PPP Pro-
of TdaR and AclT, but Asp139 in GliT (Fig. S4†). Plausibly in grammes for Project Related Personal Exchange between the
analogy to GliT-catalysed disulde bond formation, fungal National Natural Science Foundation of China and the German
oxidases utilized a similar mechanism to install disulde bonds Academic Exchange Service as well as the Deutsche For-
across a, a- or a, b-positions (Fig. 6).32 A sulydryl group in the schungsgemeinscha (DFG, German Research Foundation,
DKP-containing substrate attacks a disulde bond in the thio- INST 160/620-1).
redoxin motif (CXXC) within the oxidoreductase, creating
a transient mixed-disulde bond between the substrate and
oxidoreductase (Fig. 6A and B). In this step, FADox receives the
Notes and references
electrons at the C4a position, leading to a FAD-4a-thiol adduct.
Subsequently, it undergoes attack at the other sulydryl group
with disulde formation in the ETP alkaloid and concomitant
reduction to FADred (Fig. 6C and D). Eventually, FADred is
reoxidized by molecular oxygen instead of NADP+ with the
elimination of H2O2. We speculated that the large pocket across
the active site and FAD-binding motif of GliT as well as TdaR
and AclT50 might result in the broad substrate specicity,
making it attractive for further enzyme structure analysis and
protein engineering.37
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´
´
1171.
co-workers reported
a
biosynthetic pathway of
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4138 | Chem. Sci., 2021, 12, 4132–4138
© 2021 The Author(s). Published by the Royal Society of Chemistry