Lantibiotics are a class of peptide antibiotics that contain one or more thioether bonds. The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens1. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed by the dehydration of Ser/Thr by the lantibiotic dehydratase NisB (ref. 2). Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process3. However, the molecular mechanism by which NisB uses glutamate to catalyse dehydration remains unresolved. Here we show that this process involves glutamyl-tRNAGlu to activate Ser/Thr residues. In addition, the 2.9-Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyse the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate an unexpected role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products.
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Delves-Broughton, J., Blackburn, P., Evans, R. J. & Hugenholtz, J. Applications of the bacteriocin, nisin. Antonie van Leeuwenhoek 69, 193–202 (1996)
Sen, A. K. et al. Post-translational modification of nisin. The involvement of NisB in the dehydration process. Eur. J. Biochem. 261, 524–532 (1999)
Garg, N., Salazar-Ocampo, L. M. & van der Donk, W. A. In vitro activity of the nisin dehydratase NisB. Proc. Natl Acad. Sci. USA 110, 7258–7263 (2013)
Lubelski, J., Rink, R., Khusainov, R., Moll, G. N. & Kuipers, O. P. Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell. Mol. Life Sci. 65, 455–476 (2008)
Breukink, E. et al. Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 286, 2361–2364 (1999)
Wiedemann, I. et al. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J. Biol. Chem. 276, 1772–1779 (2001)
Hasper, H. E. et al. A new mechanism of antibiotic action. Science 313, 1636–1637 (2006)
Knerr, P. J. & van der Donk, W. A. Discovery, biosynthesis, and engineering of lantipeptides. Annu. Rev. Biochem. 81, 479–505 (2012)
Kaletta, C. & Entian, K. D. Nisin, a peptide antibiotic: cloning and sequencing of the nisA gene and posttranslational processing of its peptide product. J. Bacteriol. 171, 1597–1601 (1989)
Li, B. et al. Structure and mechanism of the lantibiotic cyclase involved in nisin biosynthesis. Science 311, 1464–1467 (2006)
van der Meer, J. R. et al. Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis. J. Bacteriol. 175, 2578–2588 (1993)
Schnell, N. et al. Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature 333, 276–278 (1988)
Li, C. & Kelly, W. L. Recent advances in thiopeptide antibiotic biosynthesis. Nat. Prod. Rep. 27, 153–164 (2010)
Shazman, S. & Mandel-Gutfreund, Y. Classifying RNA-binding proteins based on electrostatic properties. PLoS Comput. Biol. 4, e1000146 (2008)
Koehnke, J. et al. The cyanobactin heterocyclase enzyme: a processive adenylase that operates with a defined order of reaction. Angew. Chem. Int. Ed. 52, 13991–13996 (2013)
Sardar, D., Pierce, E., McIntosh, J. A. & Schmidt, E. W. Recognition sequences and substrate evolution in cyanobactin biosynthesis. ACS Synth. Biol. http://dx.doi.org/10.1021/sb500019b (13 March 2014)
Marques, J. C. et al. Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein. J. Biol. Chem. 286, 18331–18343 (2011)
Mavaro, A. et al. Substrate recognition and specificity of NisB, the lantibiotic dehydratase involved in nisin biosynthesis. J. Biol. Chem. 286, 30552–30560 (2011)
Plat, A., Kluskens, L. D., Kuipers, A., Rink, R. & Moll, G. N. Requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage. Appl. Environ. Microbiol. 77, 604–611 (2011)
Khusainov, R., Heils, R., Lubelski, J., Moll, G. N. & Kuipers, O. P. Determining sites of interaction between prenisin and its modification enzymes NisB and NisC. Mol. Microbiol. 82, 706–718 (2011)
Lubelski, J., Khusainov, R. & Kuipers, O. P. Directionality and coordination of dehydration and ring formation during biosynthesis of the lantibiotic nisin. J. Biol. Chem. 284, 25962–25972 (2009)
Zhang, Q. et al. Structural investigation of ribosomally synthesized natural products by hypothetical structure enumeration and evaluation using tandem MS. Proc. Natl Acad. Sci. USA 111, 12031–12036 (2014)
Lubelski, J., Overkamp, W., Kluskens, L. D., Moll, G. N. & Kuipers, O. P. Influence of shifting positions of Ser, Thr, and Cys residues in prenisin on the efficiency of modification reactions and on the antimicrobial activities of the modified prepeptides. Appl. Environ. Microbiol. 74, 4680–4685 (2008)
Li, J. et al. ThioFinder: a web-based tool for the identification of thiopeptide gene clusters in DNA sequences. PLoS ONE 7, e45878 (2012)
Garg, R. P., Qian, X. L., Alemany, L. B., Moran, S. & Parry, R. J. Investigations of valanimycin biosynthesis: elucidation of the role of seryl-tRNA. Proc. Natl Acad. Sci. USA 105, 6543–6547 (2008)
Zhang, W., Ntai, I., Kelleher, N. L. & Walsh, C. T. tRNA-dependent peptide bond formation by the transferase PacB in biosynthesis of the pacidamycin group of pentapeptidyl nucleoside antibiotics. Proc. Natl Acad. Sci. USA 108, 12249–12253 (2011)
Bougioukou, D. J., Mukherjee, S. & van der Donk, W. A. Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway. Proc. Natl Acad. Sci. USA 110, 10952–10957 (2013)
Gondry, M. et al. Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes. Nature Chem. Biol. 5, 414–420 (2009)
Francklyn, C. S. & Minajigi, A. tRNA as an active chemical scaffold for diverse chemical transformations. FEBS Lett. 584, 366–375 (2010)
Phizicky, E. M. & Hopper, A. K. tRNA biology charges to the front. Genes Dev. 24, 1832–1860 (2010)
Li, B., Cooper, L. E. & van der Donk, W. A. In vitro studies of lantibiotic biosynthesis. Methods Enzymol. 458, 533–558 (2009)
Kigawa, T. et al. Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J. Struct. Funct. Genomics 5, 63–68 (2004)
Sherlin, L. D. et al. Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization. RNA 7, 1671–1678 (2001)
Rio, D. C., Ares, M. J., Hannon, G. J. & Nilsen, T. W. RNA: A laboratory Manual 216–219 (Cold Spring Harbor Laboratory Press, 2011)
Walker, S. E. & Fredrick, K. Preparation and evaluation of acylated tRNAs. Methods 44, 81–86 (2008)
Janssen, B. D., Diner, E. J. & Hayes, C. S. Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis. Methods Mol. Biol. 905, 291–309 (2012)
Walter, T. S. et al. Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14, 1617–1622 (2006)
Otwinowski, Z., Borek, D., Majewski, W. & Minor, W. Multiparametric scaling of diffraction intensities. Acta Crystallogr. A 59, 228–234 (2003)
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)
Thorn, A. & Sheldrick, G. M. Extending molecular-replacement solutions with SHELXE. Acta Crystallogr. D 69, 2251–2256 (2013)
Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8, 91 (2008)
Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230 (2014)
Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011)
Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 3150–3152 (2012)
Simossis, V. A. & Heringa, J. PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information. Nucleic Acids Res. 33, W289–W294 (2005)
Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574 (2003)
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004)
We thank K. Brister and colleagues for facilitating data collection at LS-CAT (Argonne National Labs, Illinois). This work was supported by grants from the National Institutes of Health (NIH) (R01 GM 058822 to W.A.v.d.D., and RO1 GM079038 to S.K.N.). M.A.O. was supported partly by a National Institute of General Medical Sciences (NIGMS)–NIH Chemistry–Biology Interface Training Grant (5T32-GM070421) and by the Ford Foundation. Y.H. was supported partly by a Lowell P. Hager fellowship from the Department of Biochemistry. The Bruker UltrafleXtreme MALDI–TOF/TOF mass spectrometer was purchased in part with a grant from the NIH (S10 RR027109 A). The content of this work is solely the responsibility of the authors and do not necessarily represent the official views of the NIH or Ford Foundation.
The authors declare no competing financial interests.
Extended data figures and tables
a, Anion exchange chromatogram of E. coli cell extract. Fractionation was monitored at 280 nm (blue) and the sample was eluted with a NaCl gradient (red). Two peaks were observed at retention volumes of 7.3 and 10.3 ml, respectively. b–f, MALDI–TOF mass spectra of His6–NisA after in vitro reaction with His6–NisB, ATP and Glu, in the presence of the 10.3-ml fraction (b), the 7.3-ml fraction (c), E. coli cell extract (d), and E. coli cell extract treated with DNase (e) or RNase (f). Numbers above the peaks correspond to the number of dehydrations of His6–NisA after incubation with His6–NisB (b–f). M, unmodified His6–NisA (m/z 7,992, calc. m/z 7,996); 3, threefold dehydrated His6–NisA (m/z 7,941, calc. m/z 7,942); 5, fivefold dehydrated His6–NisA (m/z 7,905, calc. m/z 7,906); 7, sevenfold dehydrated His6–NisA (m/z 7,870, calc. m/z 7,870). Assays were performed in duplicate.
a, Glutamylation of in vitro transcribed E. coli tRNAGlu by purified E. coli GluRS using l-[U-14C]glutamic acid was analysed by gel electrophoresis. The gel was stained with methylene blue (top), dried, exposed to a phosphorimager screen, and scanned (bottom). Thus, recombinant purified GluRS is capable of aminoacylating E. coli tRNAGlu lacking post-transcriptional modifications. The gel was cropped for visualization purposes. b–d, MALDI–TOF mass spectra of the precursor peptide His6–NisA after incubation with His6–NisB, glutamyl-tRNAGlu and ATP (b), His6–NisB and glutamyl-tRNAGlu (c), and glutamyl-tRNAGlu in the absence of His6–NisB (d). Numbers above the mass spectra correspond to the number of dehydrations of His6–NisA after incubation with His6–NisB. M, unmodified His6–NisA (m/z 7,995, calc. m/z 7,996); 2, twofold dehydrated His6–NisA (m/z 7,959, calc. m/z 7,960); 4, fourfold dehydrated His6–NisA (m/z 7,924, calc. m/z 7,924). Dehydration assays were performed in duplicate.
Extended Data Figure 3 His6–NisB-catalysed dehydration of His6–NisA in the presence of L. lactis HP RNA and GluRS.
MALDI–TOF MS analysis of His6–NisA after incubation with His6–NisB, L. lactis His6–GluRS, RNA isolated from L. lactis, glutamate and ATP (a), His6–NisB, L. lactis His6–GluRS and RNA, and ATP (b), His6–NisB, L. lactis His6–GluRS, Glu and ATP (c), His6–NisB, L. lactis RNA, Glu and ATP (d), and L. lactis His6–GluRS and RNA, Glu and ATP (e). Numbers above the mass spectra correspond to the number of dehydrations of His6–NisA after incubation with His6–NisB. M, unmodified His6–NisA (m/z 7,997, calc. m/z 7,996); 5, fivefold dehydrated His6–NisA (m/z 7,906, calc. m/z 7,906); 7, sevenfold dehydrated His6–NisA (m/z 7,870, calc. m/z 7,870); 8, eightfold dehydrated His6–NisA (m/z 7,853, calc. m/z 7,852). Dehydration assays were performed in duplicate.
Extended Data Figure 4 Surface representation, structural homology, and model for tRNA engagement by NisB.
a, The NisB homodimer is shown with one monomer coloured in gold (glutamylation domain) and blue (glutamate elimination domain); the other monomer is coloured pink (glutamylation domain) and green (glutamate elimination domain). The NisA peptide is shown as spheres. b, Transparent cartoon representation of the dimer showing the clustering of the residues important for glutamylation (in the pink domain) and glutamate elimination (in the green domain) represented as yellow sticks. c, Calculated electrostatic potential mapped onto the NisB surface showing the basic patch (blue) that probably engages the glutamyl-tRNAGlu. The NisA peptide is shown in yellow. d, tRNAGlu-NisB binding model with the NisB glutamylation domain in pink and the elimination domain in green. The leader peptide is shown in a yellow ball-and-stick representation. The double-stranded RNA-binding protein A complexed with its cognate RNA (PDB 1DI2) was used to derive a NisB docking pose for binding to bacterial tRNAGlu (T. thermophilus tRNA taken from PDB 1N78). The model results in the placement of the aminoacylated CCA terminus in the vicinity of residues that have been shown to be important for glutamylation activity. e, Domains within the overall structure of NisB that share notable homology are shown for the leader-peptide-binding site (gold) and the site for glutamate elimination (cyan). Structures that are related by homology are shown adjacent to the respective domains.
a, Diagrammatic representation of the LsrG protein (PDB 3QMQ). Residues involved in LsrG catalysis are shown as sticks. The putative LsrG active site, located between the α-helices and the β-sheet, was proposed on the basis of the presence of an unidentified ligand17. Mutations of residues in the proposed active site of LsrG demonstrated their importance in activity, but no functions were assigned to individual residues17. b, Diagrammatic representation of the NisB glutamate elimination domain. The segment with structural homology to LsrG is coloured light blue; the remainder of the elimination domain is coloured yellow. Residues important for glutamate elimination or dehydration3 are shown as sticks. c, Residues important for glutamate elimination and net dehydration delineate a putative active site for glutamate elimination. Residues Arg 786, Arg 826 and His 961 are important for glutamate elimination3, and Glu 823 has previously been shown to be partly important for dehydration3. The importance of Arg 784 in the glutamate elimination step was determined in this study. d, MALDI–TOF MS analysis of His6–NisA purified after coexpression with His6–NisB-R784A. The presence of multiple glutamylated intermediates demonstrates that Arg 784 is important for glutamate elimination and not for glutamylation. The designations 1Glu and 2Glu above the peaks indicate the number of glutamate adducts on the family of peaks, with the number shown below indicating the additional number of dehydrations for each peak.
Extended Data Figure 6 NisB-catalysed glutamate elimination of glutamylated NisA core peptide, laser-induced deamination of full-length precursor peptide NisA, and sequence analysis of the glutamate elimination domain in NisB.
a, b, MALDI–TOF MS analysis of glutamylated NisA core peptide before (a) and after (b) incubation with His6–NisB. The data show that the leader peptide is not required for NisB-catalysed glutamate elimination. Numbers above correspond to the number of glutamate adducts or dehydrations of NisA core peptide. M, NisA core peptide (m/z 3,499, calc. m/z 3,498); 1, onefold dehydrated NisA core peptide (m/z 3,481, calc. m/z 3,480); 4, fourfold dehydrated NisA core peptide (m/z 3,427, calc. m/z 3,426); 1Glu-1, NisA core peptide after formation of one glutamate adduct and one dehydration (m/z 3,611, calc. m/z 3,609); 1Glu-2, NisA core peptide after formation of one glutamate adduct and two dehydrations (m/z 3,593, calc. m/z 3,591); 2Glu-1, NisA core peptide after formation of two glutamate adducts and one dehydration (m/z 3,740, calc. m/z 3,738); 2Glu-2, NisA core peptide after formation of two glutamate adducts and two dehydrations (m/z 3,722, calc. m/z 3,720). c, MALDI–TOF MS in reflective mode of the precursor peptide NisA. d, MALDI–TOF MS in reflective mode of NisA core peptide after treatment with the protease ArgC. ArgC cleaves after Arg −1 in the leader peptide of NisA (Fig. 1a). MALDI–TOF MS analysis of the precursor peptide NisA in reflective mode caused the appearance of smaller shoulder peaks next to the parent mass. These shoulder peaks correspond to laser-induced deamination of the parent mass and are observed only for high-molecular-mass peptides (>6 kDa). The shoulder peaks were not observed after proteolytic removal of the leader peptide, confirming that they were not the result of dehydrations. e, Sequence alignment of selected elimination domains (SpaB_C PFAM family) present in full LanBs (NisB and SpaB), thiopeptide dehydratases (NocD, SioK and CltF) and putative24 thiopeptide Diels–Alderases (NocO, SioL and CltG). Residues involved in glutamate elimination (red) are conserved in full LanBs and thiopeptide dehydratases but not in the putative Diels–Alderases.
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Ortega, M., Hao, Y., Zhang, Q. et al. Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB. Nature 517, 509–512 (2015). https://doi.org/10.1038/nature13888
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