[en] N-methyltransferase (NMT)-catalyzed methylation at the termini of nonribosomal peptides (NRPs) has rarely been reported. Here, we discover a fungal NMT LcsG for the iterative terminal N-methylation of a family of NRPs, leucinostatins. Gene deletion results suggest that LcsG is essential for leucinostatins methylation. Results from in vitro assays and HRESI-MS-MS analysis reveal the methylation sites as NH2, NHCH3 and N(CH3)2 in the C-terminus of various leucinostatins. LcsG catalysis yields new lipopeptides, some of which demonstrate effective antibiotic properties against the human pathogen Cryptococcus neoformans and the plant pathogen Phytophthora infestans. Multiple sequence alignments and site-directed mutagenesis of LcsG indicate the presence of a highly conserved SAM-binding pocket, along with two possible active site residues (D368 and D395). Molecular dynamics simulations show that the targeted N can dock between these two residues. Thus, this study suggests a method for increasing the variety of natural bioactivity of NPRs and a possible catalytic mechanism underlying the N-methylation of NRPs.
Disciplines :
Agriculture & agronomy
Author, co-author :
Li, Zixin ✱; Université de Liège - ULiège > TERRA Research Centre ; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Jiao, Yang ✱; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Ling, Jian; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Zhao, Jianlong; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Yang, Yuhong; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Mao, Zhenchuan; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China
Zhou, Kaixiang; Center for Advanced Materials Research, Advanced Institute of Natural Sciences, Beijing Normal University at Zhuhai, Zhuhai, 519087, China
Wang, Wenzhao; State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China
Xie, Bingyan ; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. xiebingyan@caas.cn
Li, Yan ; State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 100081, Beijing, China. liyan05@caas.cn
✱ These authors have contributed equally to this work.
Language :
English
Title :
Characterization of a methyltransferase for iterative N-methylation at the leucinostatin termini in Purpureocillium lilacinum.
R.A. Cacho W. Jiang Y.H. Chooi C.T. Walsh Y. Tang Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440 J. Am. Chem. Soc. 2012 134 16781 16790 1:CAS:528:DC%2BC38Xhtl2ls7jO 22998630 3482383
G. Bills et al. New insights into the echinocandins and other fungal non-ribosomal peptides and peptaibiotics Nat. Prod. Rep. 2014 31 1348 1375 1:CAS:528:DC%2BC2cXhsVeku7jK 25156669
X. Yang et al. Cyclosporine biosynthesis in Tolypocladium inflatum benefits fungal adaptation to the environment mBio 2018 9 e01211 e01218 1:CAS:528:DC%2BC1MXht12msbnK 30279281 6168864
Y. Xu et al. Biosynthesis of the cyclooligomer depsipeptide beauvericin, a virulence factor of the entomopathogenic fungus Beauveria bassiana Chem. Biol. 2008 15 898 907 1:CAS:528:DC%2BD1cXhtFCnsr3O 18804027
J.G. Beck et al. Intestinal permeability of cyclic peptides: common key backbone motifs identified J. Am. Chem. Soc. 2012 134 12125 12133 1:CAS:528:DC%2BC38Xpt1Cisbw%3D 22737969
D. Schwarzer R. Finking M.A. Marahiel Nonribosomal peptides: from genes to products Nat. Prod. Rep. 2003 20 275 287 1:CAS:528:DC%2BD3sXls1Klur8%3D 12828367
T. Velkov et al. Characterization of the N-Methyltransferase activities of the multifunctional polypeptide cyclosporin synthetase Chem. Biol. 2011 18 464 475 1:CAS:528:DC%2BC3MXltFart7s%3D 21513883
J. Lee et al. Structural and functional insight into an unexpectedly selective N-methyltransferase involved in plantazolicin biosynthesis Proc. Natl Acad. Sci. 2013 110 12954 12959 1:CAS:528:DC%2BC3sXhsVShurfI 23878226 3740862
J. Scherkenbeck A. Harder A. Plant H. Dyker PF1022A—a novel anthelmintic cyclooctadepsipeptide. Modification and exchange of the N-methyl leucine residues Bioorg. Med. Chem. Lett. 1998 8 1035 1040 1:CAS:528:DyaK1cXjtlSntLg%3D 9871703
A.F. Räder F. Reichart M. Weinmüller H. Kessler Improving oral bioavailability of cyclic peptides by N-methylation Bioorg. Med. Chem. 2018 26 2766 2773 28886995
W. Weckwerth et al. Biosynthesis of PF1022A and related cyclooctadepsipeptides J. Biol. Chem. 2000 275 17909 17915 1:CAS:528:DC%2BD3cXktFakurY%3D 10751395
S. Mori et al. Structural basis for backbone N-methylation by an interrupted adenylation domain Nat. Chem. Biol. 2018 14 428 430 1:CAS:528:DC%2BC1cXosFGmt7g%3D 29556104
F. Xu et al. Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase J. Biol. Eng. 2019 13 1 14
K.M. de Mattos-Shipley et al. The cycloaspeptides: uncovering a new model for methylated nonribosomal peptide biosynthesis Chem. Sci. 2018 9 4109 4117 29780540 5941284
A. Sandmann F. Sasse R. Müller Identification and analysis of the core biosynthetic machinery of tubulysin, a potent cytotoxin with potential anticancer activity Chem. Biol. 2004 11 1071 1079 1:CAS:528:DC%2BD2cXntVarurg%3D 15324808
T. Arai Y. Mikami K. Fukushima T. Utsumi K. Yazawa A new antibiotic, leucinostatin, derived from Penicillium lilacinum J. Antibiot. 1973 26 157 161 1:CAS:528:DyaE3sXktVektbY%3D
K. Fukushima T. Arai Y. Mori M. Tsuboi M. Suzuki Studies on peptide antibiotics, leucinostatins I. Separation, physico-chemical properties and biological activities of leucinostatins A and B J. Antibiot. 1983 36 1606 1612 1:CAS:528:DyaL2cXpvFOhsw%3D%3D
M. Kawada et al. Leucinostatin A inhibits prostate cancer growth through reduction of insulin–like growth factor–I expression in prostate stromal cells Int. J. Cancer 2010 126 810 818 1:CAS:528:DC%2BD1MXhs1SqsLjO 19795463
Y.S. Kil A.L. Risinger C.L. Petersen S.L. Mooberry R.H. Cichewicz Leucinostatins from Ophiocordyceps spp. and Purpureocillium spp. demonstrate selective antiproliferative effects in cells representing the luminal androgen receptor subtype of triple negative breast cancer J. Nat. Prod. 2020 83 2010 2024 1:CAS:528:DC%2BB3cXhtV2jtbbL 32510949 7704123
M. Brand et al. Antiprotozoal structure–activity relationships of synthetic leucinostatin derivatives and elucidation of their mode of action Angew. Chem. Int. Ed. 2021 60 15613 15621 1:CAS:528:DC%2BB3MXhtFGls7jN
A. Shima K. Fukushima T. Arai H. Terada Dual inhibitory effects of the peptide antibiotics leucinostatins on oxidative phosphorylation in mitochondria Cell Struct. Funct. 1990 15 53 58 1:CAS:528:DyaK3cXitFSlsro%3D 2140298
I. Momose et al. Leucinostatin Y: a peptaibiotic produced by the entomoparasitic fungus Purpureocillium lilacinum 40-H-28 J. Nat. Prod. 2019 82 1120 1127 1:CAS:528:DC%2BC1MXnvF2lurg%3D 31017786
A.F.C. Martinez L.A.B. Moraes Liquid chromatography-tandem mass spectrometry characterization of five new leucinostatins produced by Paecilomyces lilacinus CG–189 J. Antibiot. 2015 68 178 184 1:CAS:528:DC%2BC2MXlsVKlsbk%3D
A. Isogai J. Nakayama S. Takayama A. Kusai A. Suzuki Structural elucidation of minor components of peptidyl antibiotic P168s (leucinostatins) by tandem mass spectrometry Biosci. Biotechnol. Biochem. 1992 56 1079 1085 1:CAS:528:DyaK3sXit12ktb8%3D 1368827
G. Wang et al. Biosynthesis of antibiotic leucinostatins in bio-control fungus Purpureocillium lilacinum and their inhibition on Phytophthora revealed by genome mining PLoS Pathog. 2016 12 e1005685 27416025 4946873
A.S. Urquhart J. Hu Y.H. Chooi A. Idnurm The fungal gene cluster for biosynthesis of the antibacterial agent viriditoxin Fungal Biol. Biotechnol. 2019 6 1 13
Y. Mikami et al. Paecilotoxin production in clinical or terrestrial isolates of Paecilomyces lilacinus strains Mycopathol 1989 108 195 199 1:CAS:528:DyaK3cXhtFGns7g%3D
Y. Mori M. Suzuki K. Fukushima T. Arai Structure of leucinostatin B, an uncoupler on mitochondria J. Antibiot. 1983 36 1084 1086 1:CAS:528:DyaL2cXit12qtA%3D%3D
R. Gessmann H. Brückner A. Berg K. Petratos The crystal structure of the lipoaminopeptaibol helioferin, an antibiotic peptide from Mycogone rosea Acta Crystallogr. D Struct. Biol. 2018 74 315 320 1:CAS:528:DC%2BC1cXnt1KhsLY%3D 29652258
Corso, G., Stärk, H., Jing, B. & Jaakkola, T. Diffdock: Diffusion steps, twists, and turns for molecular docking. arXiv https://doi.org/10.48550/arXiv.2210.01776 (2022).
S.A. Newmister et al. Unveiling sequential late-stage methyltransferase reactions in the meleagrin/oxaline biosynthetic pathway Org. Biomol. Chem. 2018 16 6450 6459 1:CAS:528:DC%2BC1cXhsFahs7zF 30141817 6134404
N. Mahmoodi R.K. Harijan V.L. Schramm Transition-state analogues of phenylethanolamine N-methyltransferase J. Am. Chem. Soc. 2020 142 14222 14233 1:CAS:528:DC%2BB3cXhsVCmurzF 32702980 7558223
Q.Q. Hou J.H. Wang J. Gao Y.J. Liu C.B. Liu QM/MM studies on the catalytic mechanism of phenylethanolamine N-methyltransferase Biochim. Biophys. Acta Proteins Proteom. 2012 1824 533 541 1:CAS:528:DC%2BC38Xkt1Ggtrs%3D
A. Vit L. Misson W. Blankenfeldt F.P. Seebeck Ergothioneine biosynthetic methyltransferase EgtD reveals the structural basis of aromatic amino acid betaine biosynthesis ChemBioChem 2015 16 119 125 1:CAS:528:DC%2BC2cXhvFantLnK 25404173
S.G. Lee Y. Kim T.D. Alpert A. Nagata J.M. Jez Structure and reaction mechanism of phosphoethanolamine methyltransferase from the malaria parasite Plasmodium falciparum: an antiparasitic drug target J. Biol. Chem. 2012 287 1426 1434 1:CAS:528:DC%2BC38XjslyksQ%3D%3D 22117061
S.B. Raman B. Rathinasabapathi β-Alanine N-methyltransferase of Limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectant β-alanine betaine Plant Physiol. 2003 132 1642 1651 1:CAS:528:DC%2BD3sXlsFGhu7c%3D 12857843 167101
C.E. Schaner Tooley et al. NRMT is an α-N-methyltransferase that methylates RCC1 and retinoblastoma protein Nature 2010 466 1125 1128 1:CAS:528:DC%2BC3cXptl2rsr8%3D
J. Fricke F. Blei D. Hoffmeister Enzymatic synthesis of psilocybin Angew. Chem. Int. Ed. 2017 56 12352 12355 1:CAS:528:DC%2BC2sXhtlOrt73L
C. Tongsook et al. Structural and kinetic studies on RosA, the enzyme catalysing the methylation of 8-demethyl-8-amino-d-riboflavin to the antibiotic roseoflavin FEBS J. 2016 283 1531 1549 1:CAS:528:DC%2BC28Xkt1GgtLo%3D 26913589 4982073
P. Daniel-Ivad K.S. Ryan Structure of methyltransferase RedM that forms the dimethylpyrrolinium of the bisindole reductasporine J. Biol. Chem. 2024 300 105520 1:CAS:528:DC%2BB3sXis12mur7M 38042494
W. Zhang et al. Identifying the minimal enzymes required for anhydrotetracycline biosynthesis J. Am. Chem. Soc. 2008 130 6068 6069 1:CAS:528:DC%2BD1cXkvFShur0%3D 18422316
K.A. Larsson I. Zetterlund G. Delp L.M. Jonsson N-Methyltransferase involved in gramine biosynthesis in barley: cloning and characterization Phytochemistry 2006 67 2002 2008 1:CAS:528:DC%2BD28XptFCgt7Y%3D 16930646
K.J. Molohon et al. Structure determination and interception of biosynthetic intermediates for the plantazolicin class of highly discriminating antibiotics ACS Chem. Biol. 2011 6 1307 1313 1:CAS:528:DC%2BC3MXht1KktbzK 21950656 3241860
A. Jansson et al. Aclacinomycin 10-hydroxylase is a novel substrate-assisted hydroxylase requiring S-adenosyl-L-methionine as a cofactor J. Biol. Chem. 2005 280 3636 3644 1:CAS:528:DC%2BD2MXovVGgug%3D%3D 15548527
Y. Cai et al. Structural basis for stereoselective dehydration and hydrogen-bonding catalysis by the SAM-dependent pericyclase LepI Nat. Chem. 2019 11 812 820 1:CAS:528:DC%2BC1MXhsVWktb7I 31332284 6708486
C. Zubieta X.Z. He R.A. Dixon J.P. Noel Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases Nat. Struct. Biol. 2001 8 271 279 1:CAS:528:DC%2BD3MXhs1Khs7g%3D 11224575
S. Singh et al. Structural characterization of the mitomycin 7-O-methyltransferase Proteins Struct. Funct. Bioinf. 2011 79 2181 2188 1:CAS:528:DC%2BC3MXnt1Klt7c%3D
L. Zhang et al. Engineering the biosynthesis of fungal nonribosomal peptides Nat. Prod. Rep. 2023 40 62 88 1:CAS:528:DC%2BB38XhslOgtrfP 35796260
N. Schracke U. Linne C. Mahlert M.A. Marahiel Synthesis of linear gramicidin requires the cooperation of two independent reductases Biochemistry 2005 44 8507 8513 1:CAS:528:DC%2BD2MXktFWksL0%3D 15938641
A. Tanaka B.A. Tapper A. Popay E.J. Parker B. Scott A symbiosis expressed non-ribosomal peptide synthetase from a mutualistic fungal endophyte of perennial ryegrass confers protection to the symbiotum from insect herbivory Mol. Microbiol. 2005 57 1036 1050 1:CAS:528:DC%2BD2MXpsFCltbY%3D 16091042
H.H. Yeh et al. Resistance gene-guided genome mining: serial promoter exchanges in Aspergillus nidulans reveal the biosynthetic pathway for fellutamide B, a proteasome inhibitor ACS Chem. Biol. 2016 11 2275 2284 1:CAS:528:DC%2BC28XpsF2murs%3D 27294372 6457343
D. Berry et al. Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi Environ. Microbiol. 2019 21 928 939 1:CAS:528:DC%2BC1MXkslahtLg%3D 30452111
Y.M. Chiang et al. Development of genetic dereplication strains in Aspergillus nidulans results in the discovery of aspercryptin Angew. Chem. Int. Ed. 2016 55 1662 1665 1:CAS:528:DC%2BC2MXhvVaqt7nE
W. Li et al. Asperphenamate biosynthesis reveals a novel two-module NRPS system to synthesize amino acid esters in fungi Chem. Sci. 2018 9 2589 2594 1:CAS:528:DC%2BC1cXhsFKktro%3D 29719714 5897882
L.J. Jia et al. A linear nonribosomal octapeptide from Fusarium graminearum facilitates cell-to-cell invasion of wheat Nat. Commun. 2019 10 30804501 6389888
Y. Jiao et al. Functional genetic analysis of the leucinostatin biosynthesis transcription regulator lcsL in Purpureocillium lilacinum using CRISPR-Cas9 technology Appl. Microbiol. Biotechnol. 2019 103 6187 6194 1:CAS:528:DC%2BC1MXhtFGjurzM 31175427
R. Liu et al. Discovery of a new antifungal lipopeptaibol from Purpureocillium lilacinum using MALDI-TOF-IMS Biochem. Biophys. Res. Commun. 2020 527 689 695 1:CAS:528:DC%2BB3cXps1yjsLY%3D 32423807
Bok, J. W. & Keller, N. P. Fast and easy method for construction of plasmid vectors using modified quick-change mutagenesis. Fungal Secondary Metabolism: Methods and Protocols (eds. Keller, N. P. & Turner, G.) 163–174 (Humana Press, 2012).
K.J. Livak T.D. Schmittgen Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method Methods 2001 25 402 408 1:CAS:528:DC%2BD38XhtFelt7s%3D 11846609
N. Langlois B.K. Le Nguyen Diastereoselective syntheses of deoxydysibetaine, dysibetaine, and its 4-epimer J. Org. Chem. 2004 69 7558 7564 1:CAS:528:DC%2BD2cXnvFShsbs%3D 15497982
Y. Li et al. Emestrins: anti-Cryptococcus epipolythiodioxopiperazines from Podospora australis J. Nat. Prod. 2016 79 2357 2363 1:CAS:528:DC%2BC28Xhtl2js7bN 27557418
Bowers, K. J. et al. Scalable algorithms for molecular dynamics simulations on commodity clusters. In SC’06: Proceedings of the 2006 ACM/IEEE Conference on Supercomputing. (ed. Horner-Miller, B.) 43 (IEEE, 2006).
I. Letunic S. Khedkar P. Bork SMART: recent updates, new developments and status in 2020 Nucleic Acids Res. 2021 49 D458 D460 1:CAS:528:DC%2BB3MXntlejs7w%3D 33104802
J. Trifinopoulos L.T. Nguyen A. von Haeseler B.Q. Minh W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis Nucleic Acids Res. 2016 44 W232 W235 1:CAS:528:DC%2BC2sXhtV2isbfN 27084950 4987875
R. Zallot N. Oberg J.A. Gerlt The EFI web resource for genomic enzymology tools: leveraging protein, genome, and metagenome databases to discover novel enzymes and metabolic pathways Biochemistry 2019 58 4169 4182 1:CAS:528:DC%2BC1MXhvVent73O 31553576
