DOI: 10.1002/cbic.201200681

The C-Terminal Extended Serine Residue Is AbsolutelyRequired in Nosiheptide MaturationWeiying Liu, Min Ma, Yanjiu Xue, Nan Liu, Shuzhen Wang,* and Yijun Chen*[a]

Thiopeptides are a family of highly modified ribosomally syn-thesized peptides.[1] More than 80 thiopeptide antibiotics havebeen discovered, and their unique architectures, unprecedent-ed bioactivities, and unusual mode of action have drawn tre-mendous attention.[2] The naturally occurring thiopeptides areclassified into five series, a to e, according to the heterocycliccore, the oxidation state, and other structural features.[2c, 3] No-siheptide (1), a typical thiopeptide antibiotic, is isolated fromthe culture of Streptomyces actuosus ATCC 25421.[4] In the genecluster for the biosynthesis of 1, a single copy of the structuralgene nosM encodes a 50-aa precursor peptide containing a 37-aa leader peptide (LP) and a 13-aa core peptide (Scheme 1 A).The LP is cleaved during the maturation process, and the ma-turation pathway is mediated by at least 13 adjacently en-coded enzymes. Post-translational modifications form theframework of 1 includes a central core macrocycle (loop 1), anindolic acid ring (loop 2), and a dehydroalanine (Dha) tail(Scheme 1 A). In the maturation process of 1, the C-terminalDha tail is functionalized by NosA, which acts on intermediate2, which contains bis-Dha, by enamide dealkylation(Scheme 1 B).[5] Although NosA could also catalyze a loop 2-opened analogue in vitro,[5] whether NosA displays substratespecificity towards the C-terminal Dha residue remains un-known.

In this study, we investigated whether the extended Ser13residue in the core peptide is essential for nosiheptide matura-tion or whether it can be replaced by other amino acids. Tosubstitute the C-terminal extended Ser13, a nosM-gene-dele-tion strain (S. actuosus L1000, DnosM strain) was prepared byhomologous double crossover with a plasmid containing se-quence homology to the nosiheptide gene cluster but lackingthe nosM gene, leaving only a 6 bp scar in the chromosome(Figure S1). As anticipated, the extracts of DnosM strain failedto produce 1 compared to wild-type (Figure S2).

Subsequently, different mutated precursor peptide geneswere integrated seamlessly and in situ into the chromosomeof the DnosM strain to obtain NosM variants in S. actuosus (Fig-ure 1 A). PCR amplification and DNA sequencing confirmed theintegration of the mutated precursor peptide genes (Fig-ure 1 C). Among the mutants, threonine, with an extra methyl

group compared to serine, was first chosen for the replace-ment of Ser13 to investigate its effects on the production of 1.The S13T variant was fermented for 5 days and extracted bymethanol, then HPLC-TOF/MS was used to evaluate nosihep-tide production. Unexpectedly, this mutant lost the ability toproduce 1 with the terminal amide (Figure 2 B). AlthoughHPLC-TOF/MS analysis of WT extracts showed m/z 1222.1508[M+H]+ and m/z 1244.1326 [M+Na]+ for 1 (Figure S3 B), analy-sis of extracts of the S13T variant showed no nosiheptide; in-stead, two new analogues (6 and 7) were detected. MS spectrafor 6 showed a peak of m/z 1306.1750 [M+H]+ , correspondingto a dehydrobutyrine (Dhb) at the C terminus (Figure S4). Simi-larly, MS analysis of 7 indicated peaks of m/z 1290.1770[M+H]+ and m/z 1312.1585 [M+Na]+ , corresponding to thenon-hydroxylated analogue of 6. According to the mechanismproposed in Scheme 1 B,[5] intermediate 2, containing a Dhatail, should be transformed by NosA to generate 1 along with5 through an enamide dealkylation. However, NosA in thisstudy was unable to catalyze the substrate with a Dhb tail toyield 1, thus suggesting that Dhb blocks the enamide dealkyla-tion, especially the tautomerization step.

Next, to confirm our findings, cysteine, another structurallysimilar amino acid, was used to substitute Ser13. The S13Cmutant of NosM was obtained by using the same method asdescribed above (Figure 1). Cysteine substitution did not gen-erate any nosiheptide-related analogues (Figure 2 C). This sug-gested that the change of serine to cysteine affects the tran-scription of nosM and translation of modifying enzymes or theentire process of post-translational modification.

Glycine and alanine, the two amino acids with the smallestside chains, were also used to replace Ser13 (Figure 1 B). Aftermutagenesis and fermentation, HPLC-TOF/MS analysis of theS13G extracts showed two new analogues 8 and 9 (Figure 2 D).For 8, the peak of m/z 1280.1591 [M+H]+ matched a structurewith an additional glycine at the C terminus (Figure S5). Simi-larly, MS analysis of 9 showing a peak of m/z 1264.1642[M+H]+ indicated an non-hydroxylated analogue of 8. LikeS13G, the S13A variant produced two new analogues: 10 and11 (Figure 2 E). MS analyses of 10 and 11 (m/z 1294.1751[M+H]+ and m/z 1278.1802 [M+H]+) corresponded to ana-logues with an extra alanine at C terminus and its non-hy-droxylated compound, respectively (Figure S6). To clarify theposition of the dehydroxylation, the structures of 8 to 11 werefurther analyzed and confirmed by MS/MS (Figure S11–S14).These results indicated that neither glycine nor alanine under-go the tautomerization to serve as a substrate for NosA, andfurther demonstrated the essential role of Ser13.

To investigate whether other amino acid substitutions affectthe terminal amidation, two acidic amino acids with a potential

[a] W. Liu, M. Ma, Y. Xue, Dr. N. Liu, Dr. S. Wang, Prof. Dr. Y. ChenState Key Laboratory of Natural Medicines andLaboratory of Chemical BiologyChina Pharmaceutical University24 Tongjia Street, Nanjing, Jiangsu Province, 210009 (PRC)E-mail : shuzhenwang@cpu.edu.cn

yjchen@cpu.edu.cn

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201200681.

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for decarboxylation, aspartic acid and glutamic acid, werechosen to substitute Ser13 (Figure 1 B). After mutagenesis andfermentation, HPLC-TOF/MS spectra of the extracts showeda peak of m/z 1360.2049 [M+Na]+ and a peak of m/z1374.2217 [M+Na]+ (Figures S7 and S8); these correlate withunmodified aspartic acid and glutamic acid residues at theC terminus (calcd m/z 1360.2480 [M+Na]+ for S13D and1374.2217 [M+Na]+ for S13E), thus indicating that S13D andS13E produced analogues 12 and 13 (Figure 2 F and G,Scheme 2). Thus, the substitution of Ser13 with acidic aminoacids did not lead to any changes in the C-terminal residues,most likely through the blockage of enamide dealkylation ofthe terminal residue.

Changing the extended Ser13 of the core peptide can signif-icantly affect the maturation of nosiheptide. In particular, theS13T variant lost the ability to produce 1; however, it wasfound that this variant could generate compounds with a Dhbmoiety at the C terminus (6) and dehydroxylate Glu6 at itsg-position (7; Scheme 2). The structures of 6 and 7 were ana-lyzed and confirmed by MS/MS spectrometry (Figures S9 andS10). Because the hydroxylation might be catalyzed by NosC,

a cytochrome P450-like enzyme, the structures of 6 and thenon-alkylated intermediate were compared; the only differencewas found to be the extra methyl group on Thr13 at b-posi-tion, thus suggesting that NosC might display narrowed sub-strate specificity. Similarly, S13G and S13A produced non-hydroxylated analogues 9 and 11 (Figures 2 D and E, S12, andS14; Scheme 2). In addition, the production of 12 and 13 byS13D and S13E variants indicated that these acidic residues atthe C terminus could not be modified by the correspondingenzymes. Collectively, any change of Ser13 to a structurallyanalogous residue blocked enamide dealkylation and amideformation at the C terminus, thus indicating that only serine atthis position can be recognized by NosA to process enamidedealkylation for amidation. Moreover, the complete absence ofnosiheptide products from the non-cysteine analogues sug-gested that the functions of the extended Ser13 could bemore complicated than enamide dealkylation alone during thepost-translational modifications and product maturation. Al-though NosA can act on a loop 2-opened analogue in vitro,[5]

our results indicated that NosA cannot work on analogueswith a C-terminal residue other than serine (6–13). Due to the

Scheme 1. Biosynthesis and maturation process of nosiheptide (1). A) Core peptide processing and enamine dealkylation of intermediate 2 to afford 1. B) Pro-posed mechanism for C-terminal amidation. NosA acts on intermediate 2 for tautomerization, and nucleophilic attack by H2O generates intermediate 4. Final-ly, the acrylate unit originating from C-terminal Dha is removed to produce 1 and 5. LP and R represent the leader peptide and nosiheptide-related heterocy-clic core, respectively.

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lack of crystal structures of NosA and its homologous proteins,it is impossible to investigate the interactions between sub-strate and NosA directly. Therefore, the catalytic function andmechanism of NosA require further clarification by crystallo-graphic studies.

Currently the C-terminal functionalization is not well under-stood, and different extended residues could play distinct rolesin the post-translational processing and product maturation. Innosiheptide biosynthesis, NosA, a 151-aa protein, might be re-

sponsible for acting on the intermediate bearing a bis-Dha tailfor an enamide dealkylation to remove the acrylate unit origi-nating from the extended serine residue.[5] As nocathiacin I bio-synthesis shares a common paradigm to form the characteris-tic thiopeptide core and extended serine residue,[6] the mecha-nism of C-terminal amidation could be the same as for nosi-heptide. On the other hand, although thiostrepton possessesthe same extended precursor peptide “SCSS” as nosiheptide, itprocesses the precursor peptide in a very different fashion: byan unusual de-esterification–amidation.[7] Gene-inactivation ex-periments suggested that TsrB, a carboxylesterase, catalyzesthe hydrolysis of methyl ester to produce a carboxylic inter-mediate, and then the terminal amidation is catalyzed by TsrC,an ATP-dependent amidotransferase, to produce maturated

Figure 1. Construction of NosM variants. A) Homologous double cross-overbetween the plasmid containing nosM gene mutants and the chromosomeof S. actuosus L1000 to generate NosM variants. B) Comparison of wild-typeNosM and its mutants. Ser13 and mutated amino acid residues are indicatedin bold. C) Confirmation of nosM mutants by PCR. Lanes 1: S13T, 2: S13C,3: S13D, 4: S13E, 5: S13G, 6: S13A, 7: L1000 (DnosM), M: DNA ladders.

Figure 2. HPLC chromatograms of the extracts from wild-type (A) andmutant S. actuosus strains: B) L1210 (encoding S13T), C) L1211 (S13C),D) L1240 (S13G), E) L1241 (S13A), F) L1213 (S13D); G) L1214 (S13E). Un-marked peaks are unknown or unidentified components from the cultures.

Scheme 2. Structures of nosiheptide analogues generated by mutagenesisof Ser13.

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thiostrepton. For GE37468 and thiomuracin A, which containalanine and asparagine as their C-terminal extended residues,the C-terminal functionalization might be completely differ-ent.[1a, b] Analysis of the thiomuracin A gene cluster indicatedthat TpdH, which encodes a 444-aa protein and shares 43 %identity with peptidase M20D (GenBank number: YP_005367642), might be responsible for alanine removal, whereasGetM (35 % identity to TpdH) could be responsible for aspara-gine removal in GE37468 biosynthesis. The absence of terminalamide after substituting the extended serine for other aminoacid residues in our study provided strong evidence for thenecessity of this extension to the C-terminal functionalizationand maturation process. Because of the diverse mechanisms ofC-terminal tail formation in thiopeptides, understanding theroles of the extended amino acid residues in the C-terminalfunctionalization and the maturation of core peptide will great-ly facilitate directed biosynthesis of thiopeptides for the gener-ation of various analogues.

In conclusion, we have demonstrated that Ser13 is absolute-ly required for nosiheptide production. Mutagenesis of Ser13likely blocks enamide dealkylation of the terminal residue andaffects the entire process of post-translational modification.Further exploitation of the functions of C-extended amino acidresidues can provide new insights into the diverse mechanismof C-terminal tail formation in thiopeptides.

Experimental Section

Details of the experimental procedures and materials used in thisstudy, as well as further tables and figures are given in the Sup-porting Information.

Acknowledgements

This work was supported by the grants from the “111” Projectfrom the Ministry of Education of China and the State Adminis-tration of Foreign Export Affairs of China (no. 111-2-07), the Na-tional Key Project on Science and Technology of China (no.

2012ZX09103101-030), the National Science Foundation of China(nos. 81001378 and 81172967), the doctoral fund from the Minis-try of Education of China (No : 20110096110011), the Fundamen-tal Research Funds for the Central Universities (No : JKY2011002),and the Innovation Fund Project for Graduate Students of Jiang-su Province.

Keywords: biosynthesis · enamide dealkylation · nosiheptide ·post-translational modifications · thiopeptides

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Received: October 31, 2012Published online on February 25, 2013

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