DOI: 10.1002/cbic.201300427

Multiple Oxidative Routes towards the Maturation ofNosiheptideWeiying Liu, Yanjiu Xue, Min Ma, Shuzhen Wang, Nan Liu,* and Yijun Chen*[a]

Nosiheptide (1, Scheme 1), a secondary metabolite of Strepto-myces actuosus ATCC 25421, possesses strong activities againstGram-positive bacteria and has been used as a feed additivefor animals.[1] As a typical thiopeptide, nosiheptide contains

a macrocyclic core, an indolic acid ring, and a dehydroalanine(Dha) tail.[2] The biosynthesis of 1 was found to occur througha ribosomally synthesized and post-translationally modifiedpeptide (RiPP) system,[3] in which at least 13 post-translationalmodifications take place to convert a 13-residue precursorpeptide into the mature metabolite. Sequence analyses andvarious investigations of gene functions have clarified the ribo-somal origin of the precursor peptide, the formation of the in-dolic acid ring[3b, 4] and the maturation of the C-terminal tail.[5]

Based on the biosynthetic gene cluster of 1,[3b] nosM was con-firmed to be the precursor peptide gene by in vivo inactivationand site-directed mutagenesis. In addition, nosDEFGHO weresuggested to be responsible for framework formation becauseof the absence of intermediate production after gene inactiva-tion, and nosN and nosL were identified to be associated withthe formation of the indolic acid ring.[3b] Moreover, the matureC-terminal Dha tail was functionalized by NosA, which acts onthe bis-Dha intermediate through the process of enamine de-alkylation.[5]

Although a large picture of the biosynthetic pathway of1 has been preliminarily drawn, the detailed steps involvingcomplicated modifications remain largely elusive.[3b, 4b, 5a] De-spite the fact that oxidation occurs in most biosynthetic path-ways of microbial secondary metabolites, different oxidativeenzymes and their respective substrate specificities usually de-termine the biosynthetic direction and process.[6] Hence, theexistence of two hydroxy groups on the macrocyclic moiety of1 prompted us to exploit the oxidative steps and their relation-ship with the maturation process of 1 in the present study.Sequence alignments and phylogenetic analysis showed thatnosB and nosC in the biosynthetic gene cluster of 1 are verylikely cytochrome P450-like mono-oxygenases (Figure S1). Fur-ther comparison of the coding proteins NosB and NosC withother known P450 enzymes revealed remarkable similarity, in-cluding a conserved amino acid sequence, an O2 binding site,and a C-terminal heme-binding domain with the signature cys-teine residue for the coordination of heme (Figure S2). There-fore, the functional roles of these two putative mono-oxygen-ases were subsequently investigated to gain insights into theirinvolvements in the maturation of 1.

First, gene knockout of nosB was carried out to examine theinfluence on product formation. To avoid potential effects onthe expression of downstream gene nosA, in-frame deletionwas employed to inactivate nosB in the nosiheptide-producingstrain (wild-type S. actuosus ATCC 25421), leading to the gener-ation of mutant strain L1120 (Figure 1 A). The DnosB mutantL1120, upon HPLC-UV analysis of the fermentation extracts(Figure 1 B, lane 2), lost the capability to produce 1, and inter-mediate 2 was generated instead. Intermediate 2 was subse-quently isolated and purified from the culture broth for struc-tural determination. HR-ESI-MS analysis showed m/z 1206.1596[M+H]+ for 2 (Figure S4 A), corresponding to a molecular for-mula of C51H43N13O11S6 (m/z calcd: 1206.1602). Furthermore,MS/MS and full sets of 1D- and 2D-NMR data confirmed thestructure of 2 to be an intermediate without the hydroxygroup on Glu6 (Table S3, Figures S4, S5 B and S6), indicatingthat NosB is responsible for the hydroxylation of Glu6 at itsg-position. A single copy of nosB carrying pKL1120C was thenintroduced to L1120 to give L1120C. The resulting strain re-stored production of 1 (Figure 1 B, lane 6), demonstrating thenecessity of NosB in the maturation of 1.

To verify the catalytic function of NosB, we overexpressednosB in E. coli BL21(DE3), and the recombinant NosB was puri-fied to homogeneity (Figure S15). UV–visible spectra of NosBexhibited a peak with maximum absorbance at 418 nm. Afteraddition of Na2S2O4, followed by bubbling with CO, the maxi-mum absorption was shifted to 449 nm, showing characteris-tics of cytochrome P450-like proteins (Figure S16).[6a] Oxidation

Scheme 1. Structures of nosiheptide (1) and intermediates 2–6.

[a] W. Liu, Y. Xue, M. Ma, Dr. S. Wang, Dr. N. Liu, Prof. Dr. Y. ChenState Key Laboratory of Natural Medicines and Laboratory of ChemicalBiologyChina Pharmaceutical University24 Tongjia Street, Nanjing, Jiangsu Province, 210009 (P.R. China)E-mail : liunan0428@aliyun.com

yjchen@cpu.edu.cn

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201300427.

� 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1544 – 1547 1544

CHEMBIOCHEMCOMMUNICATIONS

of 2 by NosB was then assayed in the presence of reductaseand NADPH. Intermediate 2 was indeed converted to 1 after2 h incubation, whereas no conversion was observed withheat-treated NosB (Figure S17 B). The product of 1 was verifiedby HR-ESI-MS (Figure S17 C), further confirming the hydroxyl-ation function of NosB.

Next, an in-frame deletion of nosC was also carried outthrough homologous double crossover to generate mutantstrain L1121 (Figure 1 A). The DnosC mutant L1121, upon HPLC-UV analysis of the fermentation extracts (Figure 1 B, lane 3), lost

the capability to produce 1, but the novel intermediate 3 wasgenerated. After isolation of 3, the structure was elucidated asan intermediate without the hydroxy group at the Pyr3 posi-tion but with an extra bis-Dha tail by HR-ESI-MS, MS/MS, and1D- and 2D-NMR (Figure S10 A, Table S4, Figures S6, S7 B, andS8). To examine whether nosB gene deletion affected nosA ex-pression, a single copy of nosC carrying pKL1121C was intro-duced to L1121 to give L1121C. The resulting strain restoredthe production of 1 (Figure 1 B, lane 7), indicating that deletionof the nosC gene did not change the expression and functionof nosA.

To determine the function of NosC, we also overexpressednosC in E. coli BL21(DE3), and the recombinant NosC was puri-fied to homogeneity (Figure S18). As seen for NosB, character-istic spectra of a cytochrome P450-like protein were observedfrom NosC (Figure S19). In addition, the recombinant NosC wasable to convert its putative substrate 3 to 4 (Figure S20 B).Product 4 was identified to be an intermediate with a bis-Dha,previously obtained from inactivating nosA by mutagenesis(Figure S20 B, lane 3),[5a] which further demonstrated that NosCis a cytochrome P450-like mono-oxygenase that hydroxylatesPyr3.

Based on the results from gene deletion of nosB and nosC, itcan be concluded that NosA-catalyzed enamide dealkylation isdependent on the oxidative state of Pyr3, whereas Glu6 didnot have any effects. To address the substrate specificity of thetwo cytochrome P450-like enzymes and to clarify the oxidativeroute(s), two double gene-knockout mutants, L1122 (DnosAB)and L1123 (DnosBC), were generated using the same method.HPLC-UV analyses of the extracts of DnosAB mutant (Figure 1 B)revealed the generation of a new intermediate 5. The structureof 5 was subsequently elucidated to be an intermediate with-out hydroxylation at Glu6 but with an extra bis-Dha tail, follow-ing isolation from the culture broth of L1122 (Figure S10 A,Table S5, Figures S9, S10 B, and S11), suggesting that nosAB de-letion did not affect the hydroxylation of the Pyr3 position byNosC. On the other hand, when nosB and nosC were simulta-neously deleted (Figure 1 B), the DnosBC mutant produced in-termediate 6. Structural elucidation of 6 showed that it is anintermediate without the hydroxy groups on Glu6 and on Pyr3but with an extra bis-Dha tail (Figure S13 A, Table S6, Figures

Figure 1. Phenotypes of S. actuosus ATCC 25431 and its mutant strains, andHPLC analysis. A) The nosABC-coding region in the wild type (WT) strain isa 3061 bp DNA fragment containing the contiguous genes nosA (456 bp),nosB (1368 bp) and nosC (1227 bp). L1120 contains a 78 bp residual gene ofnosB and six bases of XbaI ; L1121 contains a 33 bp residual gene of nosCand six bases of XbaI ; L1122 contains a 21 bp residual gene of nosAB and sixbases of XbaI ; L1123 contains a 96 bp residual gene of nosBC and six basesof XbaI. The truncated protein-coding regions, after deletion, are presumedto express corresponding inactive proteins. L1120C represents the strain inwhich nosB gene is integrated into the attB site of the L1120 chromosomeby FC31-directed site-specific recombination with an ermP promoter.L1122C represents the strain in which nosC gene is integrated into the attBsite of the L1122 chromosome by FC31-directed site-specific recombinationwith an ermP promoter. B) HPLC analyses of fermentation products from WTand mutant strains. 1) WT; 2) nosB-inactivated strain L1120; 3) nosC-inactivat-ed strain L1121; 4) nosAB-inactivated strain L1122; 5) nosBC-inactivated strainL1123; 6) nosB expressed in strain L1120; 7) nosC expressed in strain L1122.

� 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1544 – 1547 1545

CHEMBIOCHEMCOMMUNICATIONS www.chembiochem.org

Sch

eme

2.P

rop

ose

do

xid

ativ

ero

ute

sfo

rth

em

atu

rati

on

of

1st

arti

ng

fro

m6.

� 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1544 – 1547 1546

CHEMBIOCHEMCOMMUNICATIONS www.chembiochem.org

S12, S13 B, and S14), indicating that deletions of nosB and nosCresulted in malfunction of NosA on the enamine dealkylation.

According to above results from gene knockout and over-expression and biochemical analyses, a hydroxylation cascadeduring the maturation of 1 was identified which involves twooxidation events catalyzed by two cytochrome P450-likemono-oxygenases. NosB acts on Glu6 at its g-position, whereasNosC acts on the Pyr3 position. These steps should occur atthe tailoring stage, after the main scaffold is formed during no-siheptide biosynthesis. Deletion of nosC completely preventedenamine dealkylation by NosA, but DnosB did not. We thusconcluded that hydroxylation at the Pyr 3 position must occurbefore cleavage of the bis-Dha tail. Therefore, three oxidativeroutes were collectively proposed for the maturation of 1(Scheme 2). However, whether intermediate 5 can be hydroxy-lated by NosB and be cleaved by NosA should be addressed toascertain the proposed routes. Subsequently, intermediate 5was evaluated as a substrate for NosB and NosA. By using re-combinant NosB and NosA, intermediate 5 was converted to 4by NosB (Figure S21) and to 2 by NosA (Figure S23), respective-ly, which closed the gap in the proposed routes.

In general, kinetic analysis can distinguish between the cata-lytic behaviors of different enzymes; however, the relativeinstability of cytochrome P450 enzymes and the strong UVabsorbance of NosB and NosC at 340 nm did not allow us todirectly or indirectly compare the kinetic differences betweenNosB and NosC against the purified intermediates. Conse-quently, we were unable to determine which route plays thedominant role or what differences the oxidative routes mightexhibit in the maturation of 1 in S. actuosus ATCC 25421, eventhough we have demonstrated the existence of three oxidativeroutes. Nevertheless, we found that NosB can tolerate threesubstrates (2, 5, and 6), and NosC can utilize two substrates (3and 6) in the present study. Given that P450 enzymes can tol-erate multiple substrates in the biosynthesis of various secon-dary metabolites,[6a, b] the entire biosynthetic process couldresult from several oxidative routes in a cooperative or syner-getic way. Among known thiopeptides, only those in the e ser-ies contain a 3-hydroxy or 3-alkoxy substituent on a 2,3,5,6-tetra-substituted pyridine central heterocyclic domain and hy-droxylation at the g-position of the glutamate reside. In thecases of glycothiohexide a, S-54832, and nocathiacin, thehydroxy groups were further attached with a glycosidic unit.[7]

Based on the high structural similarity of e series thiopeptides,their biosynthetic processes may share a common paradigm inthe formation of the characteristic macrocyclic architecture,and the modifications to produce hydroxy-pyridine and hy-droxy-glutamate may have similar mechanisms to that of nosi-heptide. Because the biosynthetic gene clusters have onlybeen reported for nosiheptide and nocathiacin in the e ser-ies,[3b, 8] further study of the biosynthetic process could facilitatethe elucidation of biosynthetic pathways of other thiopeptidesand produce novel analogues by genetic manipulation.

In conclusion, we have functionally assigned two cyto-chrome P450-like enzymes, NosB and NosC, which closely co-operate with NosA in the maturation of 1. NosB is responsiblefor the hydroxylation of Glu6 at its g-position, and NosC can

catalyze hydroxylation at the Pyr3 position. The polysubstratespecificity of NosB and NosC, in conjunction with the function-al analysis of NosA, generated three oxidative routes for thematuration of nosiheptide. The present findings have takena significant step towards the complete elucidation of the bio-synthetic pathway of nosiheptide.

Experimental Section

Details of the experimental procedures and materials used in thisstudy, as well as other tables and figures, are given in the Support-ing Information.

Acknowledgements

This work was supported by grants from the “111” Project fromthe Ministry of Education of China and the State Administrationof Foreign Export Affairs of China (no: 111-2-07), the NationalKey Project on Science and Technology of China (no:2012ZX09103101-030), the National Science Foundation of China(nos: 81001378 and No: 81172967), the Research Project of StateKey Laboratory of Natural Medicines, China Pharmaceutical Uni-versity (no: SKLNMZZ201301), the doctoral fund from the Ministryof Education of China (no: 20110096110011), the FundamentalResearch Funds for the Central Universities (no: JKY2011002), andthe Innovation Fund Project for Graduate Students of JiangsuProvince.

Keywords: biosynthesis · enamine dealkylation ·hydroxylation · nosiheptide · p450 mono-oxygenases

[1] F. Benazet, M. Cartier, J. Florent, C. Godard, G. Jung, J. Lunel, D. Mancy, C.Pascal, J. Renaut, P. Tarridec, Experientia 1980, 36, 414 – 416.

[2] a) C. Li, W. L. Kelly, Nat. Prod. Rep. 2010, 27, 153 – 164; b) J. M. Harms,D. N. Wilson, F. Schluenzen, S. R. Connell, T. Stachelhaus, Z. Zaborowska,C. M. Spahn, P. Fucini, Mol. Cell 2008, 30, 26 – 38.

[3] a) P. Arnison, M. Bibb, G. Bierbaum, A. Bowers, T. Bugni, G. Bulaj, J. Ca-marero, D. Campopiano, G. Challis, J. Clardy, P. D. Cotter, D. J. Craik, M.Dawson, E. Dittmann, S. Donadio, P. C. Dorrestein, K.-D. Entian, M. A.Fischbach, J. S. Garavelli, U. Gçransson et al. , Nat. Prod. Rep. 2013, 30,108 – 160; b) Y. Yu, L. Duan, Q. Zhang, R. Liao, Y. Ding, H. Pan, E. Wendt-Pienkowski, G. Tang, B. Shen, W. Liu, ACS Chem. Biol. 2009, 4, 855 – 864.

[4] a) Q. Zhang, Y. Li, D. Chen, Y. Yu, L. Duan, B. Shen, W. Liu, Nat. Chem. Biol.2011, 7, 154 – 160; b) Q. Zhang, D. Chen, J. Lin, R. Liao, W. Tong, Z. Xu, W.Liu, J. Biol. Chem. 2011, 286, 21287 – 21294.

[5] a) Y. Yu, H. Guo, Q. Zhang, L. Duan, Y. Ding, R. Liao, C. Lei, B. Shen, W. Liu,J. Am. Chem. Soc. 2010, 132, 16324 – 16326; b) W. Liu, M. Ma, Y. Xue, N.Liu, S. Wang, Y. Chen, ChemBioChem 2013, 14, 573 – 576.

[6] a) R. H. Lambalot, D. E. Cane, J. J. Aparicio, L. Katz, Biochemistry 1995, 34,1858 – 1866; b) D. Chen, L. Zhang, B. Pang, J. Chen, Z. Xu, I. Abe, W. Liu,J. Bacteriol. 2013, 195, 1931 – 1939; c) W. Jiang, R. A. Cacho, G. Chiou,N. K. Garg, Y. Tang, C. T. Walsh, J. Am. Chem. Soc. 2013, 135, 4457 – 4466.

[7] a) M. C. Bagley, J. W. Dale, E. A. Merritt, X. Xiong, Chem. Rev. 2005, 105,685 – 714; b) P. Northcote, M. Siegel, D. Borders, M. Lee, J. Antibiot. 1994,47, 901 – 908.

[8] a) Y. Ding, Y. Yu, H. Pan, H. Guo, Y. Li, W. Liu, Mol. BioSyst. 2010, 6, 1180 –1185; b) M. Wei, J. Deng, S. Wang, N. Liu, Y. Chen, Biotechnol. Lett. 2011,33, 585 – 591.

Received: June 30, 2013Published online on August 12, 2013

� 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 1544 – 1547 1547

CHEMBIOCHEMCOMMUNICATIONS www.chembiochem.org