Reductase of Mutanobactin Synthetase Triggers Sequential C-C Macrocyclization, C-S Bond Formation, and C-C Bond Cleavage

Macrocyclization, C −S Bond Formation, and C −C Bond Cleavage

Letter

Reductase of Mutanobactin Synthetase Triggers Sequential C−C

∥

Min Wang, Zhoujie Xie, Shoubin Tang, Ee Ling Chang, Yue Tang, Zhengyan Guo, Yinglu Cui,

Bian Wu, Tao Ye, and Yihua Chen*

Cite This: https://dx.doi.org/10.1021/acs.orglett.9b04501

Read Online

ACCESS

Metrics & More

Article Recommendations

sı Supporting Information

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

and/or a thiazepane ring are lipopeptides from Streptococcus mutans, a major causative

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.

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

macrocyclic lipohexapeptides isolated from human oral

infection of this opportunistic pathogen. Compounds 1 and

6,7

2 also exhibit immunomodulatory activities. 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) but is rare in natural

,10

products.

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).

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). 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

Figure 1. Structures of MUBs and their congeners.

bacterium Streptococcus mutans UA159. The mub biosyn-

thetic gene cluster exists in >30% of genome-sequenced S.

Received: December 16, 2019

3,4

mutans and is important for this primary causative organism

of tooth decay in surviving oxidative stress. In addition,

compounds 1−4 can blunt the hyphal formation of another

oral resident, Candida albicans, which is a critical step in the

XXXX American Chemical Society

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

Letter

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.

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 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.

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 C H N O (m/z

To test whether MubD-R is the chain oﬄoading enzyme, we

overexpressed and puriﬁed two versions of N-His -tagged

29 50

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

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

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.

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

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

identi ﬁ ed 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

7 °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

). After the macro-rings are closed by C2−C3 coupling via

Figure 3. LC−MS analysis of the MubD-R enzymatic assays using 10

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).

(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.

Compound 4, which has a 7-CH group, is just a simpliﬁed

Macrocyclization generates diverse molecular architectures

that have been observed in many clinically important

11,14,15

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

12,13

agents.

Compared with C−O and C−N bonds,

the C−C bond is rarely encountered in the closure of

2,16

-SH. The discovery of mutanolins (8 and 9) in the

macrocycles.

Characterized C−C macrocyclizations are

16−18

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

usually associated with oxidative reactions

or nucleophilic

substitutions. Herein we showed that MUBs close their

macro-rings unprecedentedly through a reduction-enabled

aldol addition. The C 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

5 in 0.5 h. HR−MS and tandem MS analyses suggested that

5 was an analog of 9 (Figures S31 and S32). A NaBH

reduction of 15 resulted in two isomers with the same

molecular formula C H O N S, excluding the possibility that

the cysteine thiol group attacked at C27 (Figure S33 ).

for lankacidin and anatoxin, except that their electrophiles

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

orcid.org/0000-0001-7138-4206

Letter

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.

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;

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

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

2,23

process.

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.

Tao Ye − Peking University Shenzhen Graduate School,

Tsinghua University Shenzhen International Graduate

School, Shenzhen, China; orcid.org/0000-0002-2780-

761

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

■

ASSOCIATED CONTENT

sı Supporting Information

■

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

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.orglett.9b04501.

(

2018YFA0901901), the NSFC grants (31570050 and

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 )

3 1 5 2 2 0 0 1 ) , t h e S h e n z h e n P e a c o c k P l a n

(KQTD2015071714043444), and the Open Project of State

Key Laboratory of Chemical Oncogenomics.

REFERENCES

■

AUTHOR INFORMATION

Corresponding Author

(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.

■

Yihua Chen − Institute of Microbiology, Chinese Academy

of Sciences, Beijing, China, and University of Chinese

Academy of Sciences, Beijing, China; orcid.org/0000-

001-9362-512X ; Email: chenyihua@im.ac.cn

(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.

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

(

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

Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

responsible for the biosynthesis of pigments involved in oxygen and

Letter

H O 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.

12) Kopp, F.; Marahiel, M. A. Macrocyclization strategies in

006, 128, 16478−16479.

polyketide and nonribosomal peptide biosynthesis. Nat. Prod. Rep.

007, 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

002, 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−

30.

17) Withall, D. M.; Haynes, S. W.; Challis, G. L. Stereochemistry

16) Tang, M. C.; Zou, Y.; Watanabe, K.; Walsh, C. T.; Tang, Y.

Oxidative cyclization in natural product biosynthesis. Chem. Rev.

017, 117, 5226−5333.

and mechanism of undecylprodigiosin oxidative carbocyclization to

streptorubin B by the Rieske oxygenase RedG. J. Am. Chem. Soc. 2015,

(

37, 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) Mejean, A.; Paci, G.; Gautier, V.; Ploux, O. Biosynthesis of

anatoxin-a and analogues (anatoxins) in cyanobacteria. Toxicon 2014,

1, 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.