pubs.acs.

org/OrgLett Letter

Reductase of Mutanobactin Synthetase Triggers Sequential C−C

Macrocyclization, C−S Bond Formation, and C−C Bond Cleavage
Min Wang,∥ Zhoujie Xie,∥ Shoubin Tang,∥ Ee Ling Chang, Yue Tang, Zhengyan Guo, Yinglu Cui,
Bian Wu, Tao Ye, and Yihua Chen*
Cite This: Org. Lett. 2020, 22, 960−964 Read Online

ACCESS Metrics & More Article Recommendations *

sı Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: Mutanobactins (MUBs) and their congeners that contain a macrocycle

and/or a thiazepane ring are lipopeptides from Streptococcus mutans, a major causative
Downloaded via NATL TAIWAN UNIV on March 4, 2020 at 00:19:29 (UTC).

agent of dental caries. Here we show that the C-terminal reductase domain of MubD
releases the lipohexapeptide intermediates in an aldehyde form, which enables a
spontaneous C−C macrocyclization. In the presence of a thiol group, the macrocyclized
MUBs can further undergo spontaneous C−S bond formation and C−C bond cleavage
to generate diverse MUB congeners.

M utanobactins (MUBs) A−D (1−4) (Figure 1) are

macrocyclic lipohexapeptides isolated from human oral
Figure 1. Structures of MUBs and their congeners.
bacterium Streptococcus mutans UA159.1,2 The mub biosyn-
thetic gene cluster exists in >30% of genome-sequenced S.
mutans3,4 and is important for this primary causative organism
of tooth decay in surviving oxidative stress.5,6 In addition,
compounds 1−4 can blunt the hyphal formation of another
oral resident, Candida albicans, which is a critical step in the
infection of this opportunistic pathogen.1,2 Compounds 1 and
2 also exhibit immunomodulatory activities.6,7 Structurally,
MUBs 1−3 are macrocyclic lipohexapeptides with a 1,4-
thiazepane ring, which has been observed in the pharmaco-
phores of several synthesized drugs (e.g., diltiazem, clentiazem,
thiazesim, and quetiapine fumarate)8 but is rare in natural
products.9,10 The 1,4-thiazepane ring is absent in 4, which has
a 7-methyl instead of a 7-SH group. Recently, Zvanych et al.
examined the metabolome of S. mutans UA159 carefully and
discovered a number of MUB congeners, including the C2-
epimer of 2 (5), MUBs I (6) and J (7) without the macrocycle,
and mutanolins A (8) and B (9) connecting an extra cysteine
at C3 (Figure 1).7
It was proposed that the biosynthesis of MUBs is initiated by
the loading of a decanoic acid to the acyl carrier protein (ACP)
domain of MubE. After seven steps of elongation catalyzed by
one polyketide synthase (PKS) module and six nonribosomal
peptide synthetase (NRPS) modules, a peptidyl carrier protein
(PCP)-tethered β-keto lauryl hexapeptidyl chain was installed
(Figure 2).5,7 To date, the linear chain offloading mechanism
remains enigmatic, let alone the macrocyclization and the other
tailoring steps. Herein we show evidence supporting that the
C-terminal reductase domain of MubD (MubD-R) releases the
linear lipohexapeptide in an active aldehyde form, which could
Received: December 16, 2019
Published: January 9, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.orglett.9b04501

960 Org. Lett. 2020, 22, 960−964
Organic Letters
Figure 2. Proposed biosynthetic pathway for MUBs and their congeners. The nonenzymatic reactions are shadowed.
trigger sequential C−C macrocyclization, C−S bond for-
mation, and C−C bond cleavage to generate MUBs and their
diverse congeners.
The bioinformatic analysis of the mub gene cluster revealed
two possible candidates for the lipohexapeptidyl chain
offloading, a standalone thioesterase (TE) encoded by mubT
and MubD-R (Figure S1). To study the function of MubT, S.
mutans T-IFD, an in-frame deletion mutant of mubT, was
constructed. When cultured under the MUB production
conditions, S. mutans T-IFD could produce MUBs, although
at a lower level compared with the wild-type strain (Figure S2),
indicating that MubT is a type-II TE with editing activity
instead of an offloading enzyme that releases the mature
lipopeptide chains.
To test whether MubD-R is the chain offloading enzyme, we
overexpressed and purified two versions of N-His6-tagged
MubD-R proteins, the MubD-R domain alone (N-MubD-R)
and MubD-R together with its adjacent thiolation domain (N-
MubD-TR) (Figure S3). The S-N-acetyl-cysteamine (SNAC)
form of the linear lipopeptide precursor of 4, β-keto lauryl
hexapeptide-SNAC (10), was chemically synthesized as a
mimic substrate of MubD-R, as depicted in Scheme S1
(Figures S4−S17). Upon incubating 10 with N-MubD-TR and
NADPH at 37 °C, a product with the same retention time as
authentic 4 was observed by LC−MS analysis (Figure 3A). Its
identity was confirmed by high-resolution mass spectrometry
(HR−MS) and tandem MS analysis (Figure S18). N-MubD-R
could also convert 10 to 4 but at a relatively lower efficiency.
Therefore, N-MubD-TR was used in the following studies.
pubs.acs.org/OrgLett Letter
When NADPH was replaced with NADH, N-MubD-TR
converted 10 to 4 at a very low rate, indicating that
NADPH is practically used by MubD-R in cells (Figure 3A).
Overall, these results suggested that MubD-R has a reductase
activity and is sufficient for the chain offloading and C−C
bond-forming macrocyclization process.
The C-terminal R domain of PKS or NRPS has never been
associated with C−C macrocyclization before. It was
envisioned that the β-keto group of MUB’s fatty acyl tail
facilitates the nucleophilic attack from C2 to C3. Therefore, we
synthesized a mimic linear substrate, butyryl hexapeptide-
SNAC (11), without the electron-withdrawing β-keto group
(Scheme S2 and Figure S19). When 11 was incubated with N-
MubD-TR and NADPH, a product with a retention time at
33.2 min was detected by LC−MS (Figure 3B). HR−MS
analysis revealed that its chemical formula is C29H50N6O7 (m/z
595.3835 [M + H]+, calcd 595.3814). This product was
identified as 12 by tandem MS analysis (Figure S20) and a
coinjection with the synthetic 12 standard (Scheme S2 and
Figure S21), which suggested that MubD-R reductively
releases the linear lipohexapeptide chain in an aldehyde form.
The aldehyde carbon is highly electrophilic and can be
coupled to the nucleophilic C2 to close the macro-rings of
MUBs. To test whether this aldol addition can take place
spontaneously or is a MubD-R catalyzed reaction, we
chemically synthesized 13 (Scheme S3 and Figures S22−
S27), the linear precursor of 4 with an aldehyde end.
Compound 13 was unstable. It tended to be oxidized to its
acid form (14) in the air (Figures S28 and S29, Scheme S1). In
961 https://dx.doi.org/10.1021/acs.orglett.9b04501
Org. Lett. 2020, 22, 960−964
Organic Letters pubs.acs.org/OrgLett
Figure 3. LC−MS analysis of the MubD-R enzymatic assays using 10
(A) or 11 (B) as a substrate.
PBS buffer (pH 7.0), it could be converted to 4 quickly at the
physiological temperature 37 °C, and the conversion was not
influenced by MubD-R (Figure S30), indicating that MubD-R
triggers the self-assembly of the macro-ring. It is reminiscent of
the macrocyclization of nostocyclopeptide, in which the C-
terminal reductase domain also releases a linear chain with an
aldehyde end that enables a nonenzymatic imine bond
formation.11
Compound 4, which has a 7-CH3 group, is just a simplified
model in investigating the formation of MUBs. In actuality, the
situation is more complex; the formation of other MUBs
involves the participation of two strong nucleophiles, C2 and
7-SH. The discovery of mutanolins (8 and 9) in the
fermentation broth of S. mutans UA159 encouraged us to
investigate the two-nucleophile situation by putting 13 in PBS
buffer with L-cysteine and monitoring this reaction mixture
with LC−MS at 37 °C. To our surprise, three sequential
spontaneous reactions were observed (Figure 4). Compound 4
was generated at the beginning and was mainly converted to
15 in 0.5 h. HR−MS and tandem MS analyses suggested that
15 was an analog of 9 (Figures S31 and S32). A NaBH4
reduction of 15 resulted in two isomers with the same
molecular formula C40H71O9N7S, excluding the possibility that
the cysteine thiol group attacked at C27 (Figure S33).
962
Letter
Figure 4. LC−MS analysis of the autoconversion of 13 in PBS buffer
(pH 7.0) with L-cysteine at 37 °C.
Compound 15 was also not stable and was slowly converted
to 16 in the mixture (Figure 4 and Figure S33). HR−MS and
tandem MS data suggested that 16 is a linear derivative with a
hemithioacetal structure (Figure S34). Compound 16 was then
oxidized in Dess−Martin reagent to generate 17 and was
identified by a coinjection with the synthetic 17 standard
(Scheme S1 and Figures S35 and S36), suggesting that the
macro-ring was reopened at C2−C3 by a retro-aldol reaction.
Guided by these results, we carefully analyzed the fermentation
broth of S. mutans UA159 for 15 and 16 by LC−MS and
observed the production of 15 in this strain (Figure S37).
We then tested whether SNAC could be used as an
alternative nucleophile in this nonenzymatic conversion
process by the incubation of 4 in PBS buffer with SNAC at
37 °C. The sequential conversion of 4 to 18 and then to 19
was detected by LC−MS and tandem MS/MS (Figures S38
and S39). Compound 19 was identified by the fact that it could
be oxidized to 10 in Dess−Martin reagent (Figure S40).
It was proposed that 20 and 21 undergo similar non-
enzymatic conversion processes to form 1−3 and 5−9 (Figure
2). After the macro-rings are closed by C2−C3 coupling via
aldol addition, nucleophiles in the cytosol (e.g., cysteine) will
compete with the 7-SH in attacking C3 to form mutanolins (8
and 9) or MUBs (1−3 and 5), respectively. The latter ones can
be converted to 6 and 7 by spontaneous retro-aldol reactions.
Macrocyclization of MUBs might confer a rigid conformation
that only allows the 7-SH to attack C3 from one side.
Therefore, only 3-(R) configuration has been discovered in the
1,4-thiazepane rings of 1−3. To prove that the macro-rings of
1−3 and 5 can be split by the spontaneous retro-aldol reaction,
2 was incubated in PBS buffer at 37 °C, and the generation of
7 was observed, as anticipated (Figures S41 and S42).
Macrocyclization generates diverse molecular architectures
that have been observed in many clinically important
agents.12,13 Compared with C−O and C−N bonds,11,14,15
the C−C bond is rarely encountered in the closure of
macrocycles.12,16 Characterized C−C macrocyclizations are
usually associated with oxidative reactions16−18 or nucleophilic
substitutions.19 Herein we showed that MUBs close their
macro-rings unprecedentedly through a reduction-enabled
aldol addition. The Cα in the vicinity of the β-keto group
(e.g., the C2 of MUBs) is a good nucleophile that is ready to
attack the electrophilic carbon atom to form a C−C bond.
MubD-R enables the C−C macrocyclization by reductively
releasing the PCP-tethered intermediates as linear lipopeptides
with an electrophilic aldehyde end (Figure 2). Macro-
cyclizations through similar nucleophilic attacks were assumed
for lankacidin20 and anatoxin,21 except that their electrophiles
https://dx.doi.org/10.1021/acs.orglett.9b04501
Org. Lett. 2020, 22, 960−964
Organic Letters pubs.acs.org/OrgLett
were imines generated by oxidative reactions. Unfortunately,
those macrocyclizations have not yet been characterized in
vitro, and it is still unknown whether their C−C bonds are
formed via spontaneous or enzymatic reactions.
The aldol addition is just the first reaction of the MubD-R
triggered three-step spontaneous process. The macrocyclized
intermediates can be further attacked at C3 by the thiol group
to generate different β-thiodiketones, including the intra-
molecular nucleophilic attack that forms the 1,4-thiazepane
ring. Actually, in several chemical syntheses from aldehydes,
diketones, and thiols, diverse β-thiodiketones were generated
via a similar two-step nucleophilic addition and thiolation
process.22,23 Here we present an interesting case to show that
such a synthesis strategy has been adopted by S. mutans to
make MUBs and mutanolins in our mouths. Moreover, S.
mutans can further convert the macrocyclized β-thiodiketones
to linear lipopeptides with a hemithioacetal end via the retro-
aldol reaction, and the third step has no precedent in the
literature. This reductase-enabled three-step relay that includes
spontaneous C−C macrocyclization, C−S bond formation,
and C−C bond cleavage in S. mutans may provide inspiration
for the chemical synthesis of macrocycles, thiazepanes, and
hemithioacetals.
In conclusion, we present an interesting example of how
microbes efficiently expand their product spectra by recruiting
chemical reactions that can take place spontaneously under the
physiological conditions. The direct products of the MUB
biosynthetic machinery in S. mutans UA159 are linear
lipohexapeptides with an aldehyde end, which can undergo
three nonenzymatic reactions sequentially to generate MUBs
and their congeners. Our results not only explain why the
MUB synthetase can generate such diverse MUBs and
mutanolins but also can guide the discovery of more MUB
congeners from S. mutans, which should be meaningful in
understanding how the mub cluster helps its S. mutans hosts in
adapting the oral environment and causing dental caries.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.9b04501.
Experimental details of the construction of the S. mutans
T-IFD mutant, enzymatic and autoconversion assays,
synthesis of substrates and standards, spectroscopic data,
■
and copies of NMR spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
Yihua Chen − Institute of Microbiology, Chinese Academy
of Sciences, Beijing, China, and University of Chinese
Academy of Sciences, Beijing, China; orcid.org/0000-
0001-9362-512X; Email: chenyihua@im.ac.cn
Other Authors
Min Wang − Institute of Microbiology, Chinese Academy
of Sciences, Beijing, China, Peking University Shenzhen
Graduate School, Tsinghua University Shenzhen
International Graduate School, Shenzhen, China, and
University of Chinese Academy of Sciences, Beijing, China
Zhoujie Xie − Institute of Microbiology, Chinese Academy
of Sciences, Beijing, China
963
Letter
Shoubin Tang − Peking University Shenzhen Graduate
School, Tsinghua University Shenzhen International
Graduate School, Shenzhen, China
Ee Ling Chang − Peking University Shenzhen Graduate
School, Tsinghua University Shenzhen International
Graduate School, Shenzhen, China
Yue Tang − Institute of Microbiology, Chinese Academy of
Sciences, Beijing, China, University of Chinese Academy of
Sciences, Beijing, China, and Peking University Shenzhen
Graduate School, Tsinghua University Shenzhen
International Graduate School, Shenzhen, China
Zhengyan Guo − Institute of Microbiology, Chinese
Academy of Sciences, Beijing, China, and University of
Chinese Academy of Sciences, Beijing, China;
orcid.org/0000-0001-7138-4206
Yinglu Cui − Institute of Microbiology, Chinese Academy
of Sciences, Beijing, China
Bian Wu − Institute of Microbiology, Chinese Academy of
Sciences, Beijing, China, and University of Chinese
Academy of Sciences, Beijing, China; orcid.org/0000-
0002-6524-2049
Tao Ye − Peking University Shenzhen Graduate School,
Tsinghua University Shenzhen International Graduate
School, Shenzhen, China; orcid.org/0000-0002-2780-
9761
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.9b04501
Author Contributions
∥
M.W., Z.X., and S.T. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Dr. Jinwei Ren, Guomin Ai, and Wenzhao Wang,
Institute of Microbiology, CAS, for technical support. This
work was supported in part by the MOST of China
(2018YFA0901901), the NSFC grants (31570050 and
31522001), the Shenzhen Peacock Plan
(KQTD2015071714043444), and the Open Project of State
Key Laboratory of Chemical Oncogenomics.
■ REFERENCES
(1) Joyner, P. M.; Liu, J.; Zhang, Z.; Merritt, J.; Qi, F.; Cichewicz, R.
H. Mutanobactin A from the human oral pathogen Streptococcus
mutans is a cross-kingdom regulator of the yeast-mycelium transition.
Org. Biomol. Chem. 2010, 8, 5486−5489.
(2) Wang, X.; Du, L.; You, J.; King, J. B.; Cichewicz, R. H. Fungal
biofilm inhibitors from a human oral microbiome-derived bacterium.
Org. Biomol. Chem. 2012, 10, 2044−2050.
(3) Liu, L.; Hao, T.; Xie, Z.; Horsman, G. P.; Chen, Y. Genome
mining unveils widespread natural product biosynthetic capacity in
human oral microbe Streptococcus mutans. Sci. Rep. 2016, 6, 37479.
(4) Hao, T.; Xie, Z.; Wang, M.; Liu, L.; Zhang, Y.; Wang, W.; Zhang,
Z.; Zhao, X.; Li, P.; Guo, Z.; Gao, S.; Lou, C.; Zhang, G.; Merritt, J.;
Horsman, G. P.; Chen, Y. An anaerobic bacterium host system for
heterologous expression of natural product biosynthetic gene clusters.
Nat. Commun. 2019, 10, 1−13.
(5) Wu, C.; Cichewicz, R.; Li, Y.; Liu, J.; Roe, B.; Ferretti, J.; Merritt,
J.; Qi, F. Genomic island TnSmu2 of Streptococcus mutans harbors a
nonribosomal peptide synthetase-polyketide synthase gene cluster
https://dx.doi.org/10.1021/acs.orglett.9b04501
Org. Lett. 2020, 22, 960−964
Organic Letters pubs.acs.org/OrgLett Letter

responsible for the biosynthesis of pigments involved in oxygen and

H2O2 tolerance. Appl. Environ. Microbiol. 2010, 76, 5815−5826.
(6) Mousa, W. K.; Athar, B.; Merwin, N. J.; Magarvey, N. A.
Antibiotics and specialized metabolites from the human microbiota.
Nat. Prod. Rep. 2017, 34, 1302−1331.
(7) Zvanych, R.; Lukenda, N.; Li, X.; Kim, J. J.; Tharmarajah, S.;
Magarvey, N. A. Systems biosynthesis of secondary metabolic
pathways within the oral human microbiome member Streptococcus
mutans. Mol. BioSyst. 2015, 11, 97−104.
(8) Bariwal, J. B.; Upadhyay, K. D.; Manvar, A. T.; Trivedi, J. C.;
Singh, J. S.; Jain, K. S.; Shah, A. K. 1,5-Benzothiazepine, a versatile
pharmacophore: a review. Eur. J. Med. Chem. 2008, 43, 2279−2290.
(9) Ohnuki, T.; Muramatsu, Y.; Miyakoshi, S.; Takatsu, T.; Inukai,
M. Studies on novel bacterial translocase I Inhibitors, A-500359s IV.
Biosynthesis of A-500359s. J. Antibiot. 2003, 56, 268−279.
(10) Shapiro, J. A.; Morrison, K. R.; Chodisetty, S. S.; Musaev, D.
G.; Wuest, W. M. Biologically inspired total synthesis of ulbactin F, an
iron-binding natural product. Org. Lett. 2018, 20, 5922−5926.
(11) Kopp, F.; Mahlert, C.; Grunewald, J.; Marahiel, M. A. Peptide
macrocyclization: the reductase of the nostocyclopeptide synthetase
triggers the self-assembly of a macrocyclic imine. J. Am. Chem. Soc.
2006, 128, 16478−16479.
(12) Kopp, F.; Marahiel, M. A. Macrocyclization strategies in
polyketide and nonribosomal peptide biosynthesis. Nat. Prod. Rep.
2007, 24, 735−749.
(13) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. The
exploration of macrocycles for drug discovery–an underexploited
structural class. Nat. Rev. Drug Discovery 2008, 7, 608−624.
(14) Kwon, H. J.; Smith, W. C.; Scharon, A. J.; Hwang, S. H.; Kurth,
M. J.; Shen, B. C-O bond formation by polyketide synthases. Science
2002, 297, 1327−1330.
(15) Gao, X.; Haynes, S. W.; Ames, B. D.; Wang, P.; Vien, L. P.;
Walsh, C. T.; Tang, Y. Cyclization of fungal nonribosomal peptides by
a terminal condensation-like domain. Nat. Chem. Biol. 2012, 8, 823−
830.
(16) Tang, M. C.; Zou, Y.; Watanabe, K.; Walsh, C. T.; Tang, Y.
Oxidative cyclization in natural product biosynthesis. Chem. Rev.
2017, 117, 5226−5333.
(17) Withall, D. M.; Haynes, S. W.; Challis, G. L. Stereochemistry
and mechanism of undecylprodigiosin oxidative carbocyclization to
streptorubin B by the Rieske oxygenase RedG. J. Am. Chem. Soc. 2015,
137, 7889−7897.
(18) Sydor, P. K.; Barry, S. M.; Odulate, O. M.; Barona-Gomez, F.;
Haynes, S. W.; Corre, C.; Song, L.; Challis, G. L. Regio- and stereo-
divergent antibiotic oxidative carbocyclizations catalysed by Rieske
oxygenase-like enzymes. Nat. Chem. 2011, 3, 388−392.
(19) Nakamura, H.; Schultz, E. E.; Balskus, E. P. A new strategy for
aromatic ring alkylation in cylindrocyclophane biosynthesis. Nat.
Chem. Biol. 2017, 13, 916−921.
(20) Arakawa, K.; Sugino, F.; Kodama, K.; Ishii, T.; Kinashi, H.
Cyclization mechanism for the synthesis of macrocyclic antibiotic
lankacidin in Streptomyces rochei. Chem. Biol. 2005, 12, 249−256.
(21) Méjean, A.; Paci, G.; Gautier, V.; Ploux, O. Biosynthesis of
anatoxin-a and analogues (anatoxins) in cyanobacteria. Toxicon 2014,
91, 15−22.
(22) Ma, X.; Zhang, M.; Wang, B.; Min, F. Microwave-assisted
synthesis of β-thiodiketone compounds by multicomponent reaction.
Phosphorus, Sulfur Silicon Relat. Elem. 2015, 190, 1108−1114.
(23) Dar, A. A.; Enjamuri, N.; Shadab, M.; Ali, N.; Khan, A. T.
Synthesis of unsymmetrical sulfides and their oxidation to sulfones to
discover potent antileishmanial agents. ACS Comb. Sci. 2015, 17 (11),
671−681.

964 https://dx.doi.org/10.1021/acs.orglett.9b04501
Org. Lett. 2020, 22, 960−964