Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum

Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum Yan Li, Zixin Li, Yang Jiao, Jian Ling, Jianlong zhao, Yuhong Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3280468/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jun, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract N- methyltransferase (NMT)-catalyzed methylations are rarely reported at nonribosomal peptides (NRPs) terminuses. Here, we discovered a fungal NMT LcsG for the iterative terminal N -methyl formation of a family of NRPs, leucinostatins. Gene deletion suggested LcsG is essential to the methylation of leucinostatins. In vitro assay and HRESI-MS-MS analysis proved the methylation sites were the NH 2 , NHCH 3 and N(CH 3 ) 2 in the C-terminal unit of various leucinostatins. Based on the protein structure predicted by artificial intelligence (AI), molecular docking, and site-directed mutagenesis, we proposed the catalytic mechanism of the LcsG-catalyzed reaction was an N atom coordinated by two negatively charged residues (Asp368, Asp395 for LcsG) towards the subsequent S N 2 methylation. These findings not only provide an approach for enriching the variety of natural bioactivity of NPRs but also deepen the insight into the catalytic mechanism of N -methylation of NRPs. Biological sciences/Microbiology/Fungi/Fungal biology Biological sciences/Molecular biology/Post-translational modifications/Methylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction N -methyltransferases (NMTs) are ubiquitous tailoring enzymes found in all kingdoms of life. NMTs often function as the modulator of signaling molecules in animal signal transduction pathways. 1–3 NMTs are also employed in the biosynthetic pathways of natural products. 4–7 The N -methylations catalyzed by NMTs contribute significantly to changing the property of natural products by modifying their structures and influencing their bioactivities. 8 In the biosynthesis of nonribosomal peptides (NRPs), NMTs often integrate into the nonribosomal peptides synthase (NRPS) as domains, while seldom acting as a freestanding enzyme. 9,10 Although the existing examples describe enzymatic mono- N -methylations in peptide bonds or side chains of NRPs, 11–13 NMT-catalyzed iterative N -methylation at the terminus of NRPs has been rarely reported. Leucinostatins are a family of lipopeptide antibiotics, derived from Purpureocillium lilacinum . 14 They exhibit a wide range of biological activities affecting multiple pathogens. 15 Furthermore, they have been studied as potential anticancer agents and potent antiprotozoal agents. 16–18 Their bioactivities are attributed to the inhibition of mitochondrial function. 19,20 At least 24 leucinostatin homologues have been isolated and characterized. 21 Their backbone chains are assembled by a NRPS with nine amino acids and an atypical amino moiety at C -terminus, linked by peptide linkages. 22 The C -terminuses of leucinostatins are methylated to varying degrees. Specifically, the C -terminus of leucinostatin C ( 1 , LeuC) is propane-1,2-diamine (PD), while that of leucinostatin B ( 2 , LeuB) and leucinostatin A ( 3 , LeuA) are protected by N -methylpropane-1,2-diamine (MPD), and N 1 , N 1 -dimethylpropane-1,2-diamine (DMPD), respectively (Fig. 1 A). However, the biosynthetic mechanisms of the diverse C -terminuses remain unknown. In this work, we identify a discrete NMT that could catalyze a unique moiety located in the terminus of a NRP, named as LcsG, from the biological control fungus P. lilacinum PLBJ-1. 23 Deleting the lcsG gene led to the disappearance of leucinostatins with methylated terminus. Subsequent in vitro enzyme activity assays and structure elucidations of products demonstrate the LcsG conducts iterative methylation at the terminal-free amines of leucinostatins. Moreover, the structure-function relationship analysis provided a probable insight into the catalytic mechanism of LcsG. Additionally, we obtained new leucinostatins from the LcsG-catalyzed reaction, which could inhibit the growth of the human pathogen Cryptococcus neoformans and the plant pathogen Phytophthora infestans . Results Identification of LcsG from Purpureocillium lilacinum Our previous studies demonstrated that the strain P. lilacinum PLBJ-1 harbors the leucinostatin biosynthetic gene cluster (BGC), 23 and the gene lcsG whose function was predicted as a methyltransferase attracted our interest (Fig. 1 B). The LcsG contained the Methyltransf_2 domain that is typical for O -methyltransferases (OMTs) (Table S1 ) and the top hit (31.65% sequence identity) of its pBLAST research was an OMT, VdtC (A0A443HJY8.1). 24 Despite this prediction, we could not find an O -methylated unit in leucinostatins. This discrepancy prompted our curiosity to analyze the function of LcsG. To figure out the function of LcsG in the leucinostatins biosynthesis, we constructed a deletion mutant (Δ lcsG ) and an overexpression mutant (OE lcsG ) of P. lilacinum PLBJ-1 (Fig. S1 -S3). Following growth on a productive medium and production extraction, LC-MS analysis suggested obvious differences between the deletion mutant and wild-type strain (WT) (Fig. 1 C), while there were no obvious differences between the overexpression mutant and WT (Fig. S3). The LC-MS analysis of WT extracts showed the m/z [M + H] + of four peaks ( 1 – 4 ) to be 1190.8133, 1218.8439, 1204.8247, and 1234.8388, respectively (Fig. S4). High-resolution electrospray ionization mass spectrometry (HRESI-MS-MS) analysis (Table S2 and Fig. S5) confirmed peaks 1 – 3 as the known LeuC, LeuB, and LeuA, respectively. HRESI-MS-MS and NMR analysis suggested peak 4 was not the known compound leucinostatin K (LeuK), but a compound derived from LeuC whose C -terminus was NH-CH 2 -CH 2 -OH or CH 2 -CH 2 -O-NH 2 and designated as leucinostatin K0 ( 4 , LeuK0) (Fig. S6-S8). A specific N -hydroxysuccinimide (NHS)-ester reaction was employed to verify the free amine in LeuK0, and then the product compound LeuK1 ( 5 ) with NHS ester labeling was detected (Fig. S9), which confirmed the structure of LueK0 ( 4 ) as shown in Fig. 1 A. The deletion of lcsG only led to the abolishment of LeuB and LeuA, suggesting the deletion blocked the formation of the methylated C -terminal amines (Fig. 1 C). Therefore, LcsG was inferred to play an essential role in the biosynthesis of the terminal amines of leucinostatins. LcsG functions as a SAM-dependent methyltransferase Sequence analysis suggested that LcsG is an S -adenosyl- L -methionine (SAM)-dependent methyltransferase. To clarify its biochemical function, we expressed lcsG in E. coli ArcticExpress (DE3) and purified the recombinant His 6 -tagged protein LcsG by nickel affinity chromatography (Fig. 2 A). The enzymatic activity was assayed using the LeuK0 ( 4 ) and LeuA ( 3 ) as the substrates, respectively. The S-adenosyl- L -homocysteine (SAH) and three new product peaks LeuK2 ( 6 ), LeuK3 ( 7 ) and LeuA0 ( 8 ) were only detected in the presence of the substrate, LcsG, and SAM (Fig. S10 and 2B). In contrast, omitting either LcsG or SAM resulted in no product formation. LC-MS analysis proved the [M + H] + of ions of 6 – 8 were 1248.8533, 1262.8711, and 1232.8595, respectively (Fig. S11). These molecular weights indicated LeuK2 ( 6 ) and LeuA0 ( 8 ) should be the methylated products of LeuK0 ( 4 ) and LeuA ( 3 ), respectively, and LeuK3 ( 7 ) should be the dimethylated product of LeuK0 ( 4 ). These results indicated that LcsG is likely a SAM-dependent methyltransferase. The kinetic activity of LcsG was analyzed to gain further insight into the methylation activity. We conducted several single-factor enzymatic assays by using the crude extract of WT as the substrate and compared the differences between the control groups and the experimental groups by LC-MS to measure the variations of each component. The time dependency of the LcsG-catalyzed reaction showed that LeuA0 ( 8 ) and LeuK3 ( 7 ) accumulated nearly linearly with time and appeared almost immediately after the reaction started. LeuB ( 2 ) and LeuK0 ( 4 ) were nearly completely consumed after the beginning of the reaction, while the amount of LeuA ( 3 ) and LeuK2 ( 6 ) increased early on, then decreased (Fig. 2 C). These results suggested that LcsG is an iterative methyltransferase, and the reaction sequences could be LeuB-LeuA-LeuA0 and LeuK0-LeuK2-LeuK3. The optimal pH and temperature for producing the final products LeuA0 and LeuK3 were determined (Fig. S12), followed by the measurements of the initial rates at substrate concentrations ranging from 0-200 µM. The initial rate data were measured by LC-MS and fitted to the Michaelis-Menten equation to derive kinetic parameters (Fig. 2 D and Fig. S13). The K cat / K m values were 65.39 s -1 M -1 and 32.94 s -1 M -1 towards LeuA ( 3 ) and LeuK0 ( 4 ), respectivelyand LeuA, respectively, and LeuK3 might be the dimethylated product of LeuK0. Characterizations of the products of LcsG-catalyzed reaction To elucidate the structures of compounds LeuK2 ( 6 ), LeuK3 ( 7 ) and LeuA0 ( 8 ), we turned to HRESI-MS-MS analysis. The m/z values of fragments of each leucinostatins were presented in Table S2. Comparisons of the MS-MS data of LeuA0 ( 8 ) with those data of LeuA ( 3 ) and LeuB ( 2 ), found that the spectrum of LeuA0 ( 8 ) showed remarkably similar fragments with LeuA ( 3 ) and LeuB ( 2 ) (Fig. S14). Specifically, they shared one same fragment whose m/z value was 1173. This ion was deduced to be the [M + H] + ion of the fragment C10, indicating the possible methylated site of the C -terminal amine. Therefore, LeuA0 ( 8 ) was concluded to be a trimethylammonium compound in which the terminal amine carried a positive charge and three methyl groups. This predicted structure is identical to a previously identified structure, 25 which was obtained by treating LeuA ( 3 ) with methyl iodide. This reaction was prepared and gave the same product as the enzymatic reaction (Fig. 3 A), confirming the structures of LeuA0 (Fig. 3 C). Combined with the molecular weight, LeuA0 ( 8 ) was assigned the molecular formula C 63 H 114 N 11 O 13 + . Similarly, treating LeuK0 ( 4 ) with methyl iodide also gave the products LeuK2 ( 6 ) and LeuK3 ( 7 ) (Fig. 3 B). According to the molecular weights of LeuK2 ( 6 ) and LeuK3 ( 7 ), their molecular formulas should be C 63 H 113 N 11 O 14 and C 64 H 115 N 11 O 14 , respectively. From the MS-MS experiment, they also presented similar fragments as LeuK0 (Fig. S15). The differences were observed in the fragments belonging to the Y type. The Y type fragments in LeuK2 ( 6 ) are + 14 mass units greater than the corresponding fragments observed in LeuK0 ( 4 ). For example, the m/z values 190 and 204 refer to [M + H] + ions of fragments Y2 in LeuK0 and LeuK2 ( 6 ), respectively. Furthermore, LeuK2 ( 6 ) and LeuK3 ( 7 ) also afforded an ion at m/z 1173 (C10) indicating the possible methylation of the terminal amine of LeuK2 ( 6 ). Additionally, LeuK2 ( 6 ) and LeuK3 ( 7 ) were new leucinostatins (Fig. 3 C). These results demonstrated that the LcsG is a NMT that could iteratively catalyze the methylation of the terminal amine of NRP. Antifungal evaluation of leucinostatins Leucinostatins are well-known antibiotics. We carried out the purification for the compounds and obtained LeuA0 ( 8 ), LeuK0 ( 4 ) and LeuK3 ( 7 ) in sufficient quantities to do the antifungal assay. We determined the inhibitory activity of these compounds against the drug resistant strain C. neoformans H99 and the plant pathogen P. infestans by agar diffusion assays. All these leucinostatins showed inhibitory effects against these pathogens (Fig. 3 D). Moreover, the anti- C. neoformans MIC value of the two methylated products, LeuA0 ( 8 , 25.8 µg/mL) and LeuK3 ( 7 , 25.8 µg/mL), were four and two times lower than those of their parent molecules, LeuA ( 3 , 102.4 µg/mL) and LeuK0 ( 4 , 51.2 µg/mL), respectively (Fig. S16), which indicated that N -methylation at the terminus of leucinostatins could improve their antifungal efficiencies. Catalytic mechanism of N -methyltransferase LcsG We then identified the catalytic residues in LcsG. Local multiple sequence alignments revealed that LcsG shared a conserved SAH/SAM binding motif. Despite many attempts, we were unable to obtain a crystal of LcsG protein suitable for X-ray crystallographic analysis. As an alternative, we employ artificial intelligence (AI) methods to approximate a model of LcsG. The overall structure of LcsG is a typical ClassI methyltransferase fold, the N -terminus appears responsible for dimerization and substrate binding and the C -terminus appears responsible for SAM binding. To unravel the structure-function relationship of LcsG, we conducted a molecular docking analysis using the predicted LcsG structure and SAH. SAH was docked into the LcsG structure model’s binding pocket and the hydrogen bonds network that mediates SAH binding was present in the final docking position. As shown in Fig. 4 A, potential hydrogen bond interactions between SAH and residues Asp296, Asp321, Asp348, and Lys363 were presented, which was consistent with the result of multiple sequence alignment (Fig. S17). The accurate stereo-structure structures of nonpeptide leucinostatins are hard to predict because they comprised seven non-standard and unusual α-amino acid residues. Based on the crystal structures of analogues helioferin A 26 and ZHAWOC6027 18 , structural model of LeuA was predicted, and docked into LcsG structure via Diffdock. 27 In the first ranked result, the N atom which would be methylated occupies the position between Asp368, Asp395 and SAH (Fig. 4 B). To verify this, we mutated Asp296, Asp321, Asp348, Lys363, Asp368, and Asp395 to Ala in LcsG (Fig. S18). Biochemical assays of these mutants were then performed using LeuA as substrate. After 1 hour incubation, all mutants showed decreases in the conversion of LeuA ( 3 ) to LeuA0 ( 8 ) (Fig. 4 C). These results were aligned with the previous results of in vitro assays and molecular docking, suggesting the binding site of SAM of LcsG in leucinostatins methylation and further supported that LcsG was a SAM-dependent methyltransferase. The reaction mechanism of OMT has been proposed in several studies. 28 In the proposed mechanism, a His/Glu dyad and an Asp residue were required. The Glu residue was placed near the His residue and activated the His residue to deprotonate the hydroxyl group in the substrate. The Asp residue was shown to interact with the substrate for improved binding. The deprotonated hydroxyl group would function as a good nucleophile to attack the SAM, which is the methyl donor, to form the O -methylation product. For NMT, similar but different mechanisms have been proposed. 29 A QM/MM study on the catalytic mechanism of phenylethanolamine NMT 30 stated that unlike OMT, a Glu residue was employed in the deprotonation step. The protonated amine in the substrate was deprotonated by a Glu residue to form a nucleophile. Then, the methyl group was transferred from the methyl-donor SAM to the deprotonated amine group. For LcsG, the mutation data and docking result indicated that the reaction mechanism of LcsG is like phenylethanolamine NMT. Unlike reactions catalyzed by OMT, a His/Glu dyad was not required. The lone electron pair of the N atom on the dimethylamine group could undergo a nucleophilic attack. This was consistent with the fact that in sequence alignment, the corresponding residue of OMT His was His367 (Fig. S19). We proposed that the protonated dimethylamine group in the leucinostatins is coordinated and deprotonated by two negatively charged residues (D368, D395). A nucleophilic attack between the dimethylamine and SAM follows and the methyl group was transferred from SAM to leucinostatins. Compared to wild-type LcsG, the LcsG-D368A and LcsG-D395A showed obvious decreases (Fig. 4 C), indicating these two residues contribute significantly to the substrate binding. Discussion NMTs are important for the biotransformation of bioactive molecules. The N -methylation can modulate the activity of signaling molecules and participate in the biosynthesis of natural products. NMTs are of great interest because the site-specific modification is crucial for the bioactivity and biosynthesis of natural products. In this study, we successfully determined the in vitro activity of the NMT, LcsG, involved in the iterative N -methylation of the unique terminal unit of leucinostatins. Few NMTs are known to iteratively transfer methyl groups to natural products. The mycobacterial NMT EgtD 31 , Plasmodium falciparum PfPMT 32 , and human NRMT ( N -terminal RCC1 methyltransferase) 1 were reported to progressively catalyze N -trimethylation of corresponding substrates. The Psilocybe NMT PsiM 33 was shown the capacity of catalyzing geminal N -dimethylation. Although the NMTs involved in the biosynthesis of plantazolicin class ribosomally synthesized and posttranslationally modified peptides (RiPPs) are responsible for the dimethylation, the corresponding monomethylated products were never detected 8,34 . Besides, the phylogenetic analysis of LcsG and all related NMTs suggested that LcsG is closely related to OMTs although it clusters with NMT (Fig. S20). NRPSs are well-known megaenzymes that consist of sequential domains. The peptide is elongated followed by released from the terminal module, including condensation domains (C T ), reductase domains (R), Dieckmann cyclase domains (D), and thioesterase domains (TE) 35 . According to the analysis of antiSMASH 2.0 and pBLAST results (Table S3), the terminal module of the NRPS in the biosynthetic gene cluster (LcsA) should be an R domain that could release the peptide from the NRPS by hydrolysis, so that the C -terminus of leucinostatins normally should be an aldehyde group 36–39 or a hydroxyl group 40–42 (Fig. S21). However, we have not seen such similar structures among the existing characterized leucinostatins. Based on these reports and the NMR results of LeuK0, we initially deduced the C -terminal of LeuK0 to be NH-CH 2 -CH 2 -OH, but this hypothesis was rejected by the subsequent NHS ester reaction and enzyme assays. For OMT OxaC and CHOMT, the key catalytic sites responsible for catalyzing the methyltransfer reaction were reported as His313 and His278, respectively. Interestingly, the corresponding residue in LcsG is Tyr according to the multiple sequence alignments. The multiple sequence alignment result suggested the corresponding residues in other reported OMTs were basic residues (His), and residues in NMTs were neutral or acidic (Fig. S22). The LcsG-catalyzed N -methyl transfer reaction is expected to occur via nucleophilic attack by the lone electron pair of terminal N of leucinostatins on the reactive sulfonium methyl group of SAM 43 . The O -methyltransfer reaction needs a base-assisted deprotonation step to generate a nucleophile 44 , and this may be the reason why OMTs share a His residue. On the contrary, the deprotonation of the N -methyltransfer reaction does not require a basic residue. In conclusion, an OMT-like enzyme from P. lilacinum , LcsG, was identified as a discrete SAM-dependent NMT which can iteratively catalyze the primary amine, secondary amine, and tertiary amine in the unique terminal unit of leucinostatins. Furthermore, one new secondary metabolite (LeuK0) and two enzymatic products (LeuK2 and LeuK3) were identified as new leucinostatins. In addition, the methylated compounds were observed to display higher antifungal activities than their parent molecules. To our knowledge, LcsG is a rare NMT that can methylate the terminal residues of NRPs. We expect that the results of this study provide deeper insights into mechanisms of N -methylation of peptides and create the possibility of engineering new methylated molecules for exploring more potent antibiotics. Methods Strains and cultural conditions The strains used in this study are listed in Supplementary Table 4. The Purpureocillium lilacinum strain PLBJ-1 (CGMCC3.17492) 23,45,46 and its transformants were cultured at 28°C in potato dextrose agar (PDA) or potato dextrose broth (PDB) with appropriate antibiotics as required. Escherichia coli Trelief 5α (Tsingke, China) was cultured at 37°C in Luria-Bertani (LB) broth with appropriate antibiotics. The E. coli ArcticExpress (DE3) (Agilent Technologies) was used for LcsG protein expression. E. coli ArcticExpress (DE3) was cultivated at 37°C in LB broth with appropriate antibiotics for growth, followed by growth at 11°C for inducing the recombinant protein. Bioinformatics analysis of LcsG and phylogenetic tree construction Sequences of MTs were downloaded from the NCBI database. Multiple sequences were aligned with MUSCLE. The phylogenetic trees were constructed using the neighbor-joining method with bootstrap support of 1000 in MEGAX. The trees were visualized by using the Interactive Tree of Life (ITOL, http://itol.embl.de/ ). The amino acid sequence of LcsG was used as a query for BLASTp analysis. DNA and RNA isolation The mycelia of PLBJ-1 and mutants were harvested via filtration. The genomic DNA was extracted using a Qiagen DNeasy Kit. The RNA was extracted using a TRIZOL reagent (Takara, Japan) following the manufacturer’s protocol. Gene cloning and plasmid construction The oligonucleotide sequences for PCR primers are listed in Supplementary Table 5. PCR reactions were performed using 2 × Phanta Max Master Mix Polymerase (P525, Vazyme Biotech Co., Ltd, China) and Q5 High-Fidelity DNA Polymerase (New England Biolabs, USA). The plasmids are listed in Supplementary Table 4. To construct the deletion cassette of lcsG , about 1 kb DNA fragments located upstream and downstream of the lcsG coding region were amplified from the gDNA of PLBJ-1, named lcsGup and lcsGdown , respectively. Two fragments and the selection marker gene neo were integrated into the Kpn I/ Bam HI-cleaved vector pKOV21 via the digestion-ligation method by using T4 DNA Ligase (Thermo Fisher Scientific, USA) to give the deletion plasmid pKOV21-ko lcsG . For the overexpression of lcsG in PLBJ-1, the lcsG gene was amplified from the cDNA of PLBJ-1. The selection marker gene neo and the terminator TrpC were amplified from the KSTNP vector. This fragment and two restriction enzyme cutting sites Pme I and Pac I were integrated into the pEASY vector by using pEASY ®-Blunt cloning Kit (TransGen Biotech, China) to obtain the intermediate vector pEASY- neoTrpC . Then the strong promoter gpdA was amplified from the PCH-sGFP vector and integrated into the Not I/ Apa I-cleaved vector pEASY- neoTrpC via the digestion-ligation method by using DNA T4 Ligase to obtain the vector pGNT. Afterwards, the lcsG gene was integrated into the Pme I/ Not I-cleaved vector pGNT by using pEASY ®-Basic Seamless Cloning and Assembly Kit (TransGen Biotech, China) to obtain the overexpression vector pGNT- lcsG . The recombinant protein LcsG expression vector pACYC- lcsG was generated by integrating the lcsG gene from the PLBJ-1 cDNA into the protein expression vector pACYCDuet-1 by using the Quick-change method 47 . The mutated LcsG protein vectors were obtained by using QuickMutation™ Site-Directed Mutagenesis Kit (D02065, Beyotime Biotechnology, China) following the manufacturer’s instructions. PEG-mediated fungal transformation The split-marker strategy was used in disrupting the lcsG gene. The DNA fragments lcsGup - ne ( lcsGup and the first half of neo ) and eo - lcsGdown (the second half of neo and lcsGdown ) were amplified from pKOV21-ko lcsG . The two fragments of neo overlapped by 667 bp. 5 µg of two DNA fragments were transformed into PLBJ-1 by the polyethylene glycol (PEG)-mediated fusion of protoplasts according to the described protocol 45 . Geneticin (G418) resistant colonies were selected after culturing on PDA at 28°C for 1 day. The candidate transformants were picked and inoculated into new PDA plates with 400 µg/mL G418 (Inalco, USA) (400 µg/mL). These transformants were verified via diagnostic PCR with primers. For the overexpression of lcsG , the plasmid pGNT- lcsG and the empty vector pGNT were transformed into PLBJ-1 to construct the overexpression and control strain, respectively. qRT-PCR analysis of lcsG overexpression strain For cDNAs synthesis, about 1 µg of DNase-treated, RNase-cleaned RNA was used as the template by using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (R312, Vazyme Biotech Co., Ltd, China). Three biological replicates were measured for each analysis of the relative expression levels. The housekeeping gene actin (Genebank number VFPBJ_07912) was used as a control. The qRT-PCR was run with ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech Co., Ltd, China) on a BIO-RAD CFX96 (BIO-RAD). The relative expression values were calculated using the 2 −ΔΔCt method 48 . Primers are listed in Table S2. Culture extraction PLBJ-1 and its mutants were cultured in PDB medium at 28°C and 220 rpm for 14 days. The fermentation was extracted with an equal volume of ethyl acetate (EtOAc) three times (each 1 hour) and EtOAc evaporated under reduced pressure. The extract was redissolved in acetonitrile (MeCN) for further experiments. Chemical methylation of LeuA and LeuK0 For chemical methylation of LeuA and LeuK0, diisopropylethylamine (15 µL) and iodomethane (55 µL) were successively added to a solution of LeuA and LeuK0 (10 mg) in dry tetrahydrofuran (THF) (0.6 mL), respectively. These mixtures were stirred at room temperature at 800 rpm for 46 hours, and volatile constituents were evaporated at room temperature 49 . For isolating LeuA0 and LeuK3, the WT and Δ lcsG mutant crude extract were used as substrates, respectively. Product purification of LeuK0, LeuA0, and LeuK3 To isolate LeuK0, the Δ lcsG mutant was inoculated into PDB medium and incubated at 28°C for 14 days. LeuA0 and LeuK3 were isolated from chemically methylated WT and Δ lcsG mutant crude extract, respectively. LeuK0, LeuA0, and LeuK3 were purified by semi-preparative HPLC from crude extracts mentioned above, respectively. The UV absorption of leucinostatins was monitored at 214 nm with the HPLC’s DAD. Samples were separated on an Agilent 1260 Infinity II HPLC system with a Kromasil 100-5-C18 column (10 mm × 250 mm), eluted with a linear gradient of 20–70% of MeCN-water for 25 min at a flow rate of 2 mL/min. The retention time of LeuK0, LeuA0, and LeuK3 was 23.8 min, 21.6 min, and 22.3 min, respectively. Structure characterization of LeuK0 The compound LeuK0 was assigned a molecular formula of C 62 H 111 N 11 O 14 on the base of HRESIMS ( m/z 1234.8383 [M + H] + ). Its ESI-MS-MS data were compared with those of previously reported leucinostatins A-C (Table S3). The spectra of these compounds showed the same fragments (from B1 to C10), which indicated that their structural feature (from B1 to C10) is the same and the C -terminal unit (C 2 H 6 NO) in LeuK0 is different. Analysis of its 13 C-NMR APT, DEPT-135, and DEPT-90 spectroscopic data (Fig. S7-S9) revealed a total of 62 carbons, including 18 methyl groups (-CH 3 ), 14 methylenes (-CH 2 ), 16 methines (-CH), and 14 sp 3 quaternary carbons. Since the same unit (from B1 to C10) already contains 18 -CH 3 , 12 -CH 2 , 16 -CH, and 14 sp 3 quaternary carbons, thus the C -terminal unit (C 2 H 6 NO) in LeuK0 was thought to be -CH 2 -CH 2 -O-NH 2 or CH 2 -CH 2 -OH. N -hydroxysuccinimide (NHS)-ester reaction 7-Methoxycoumarin-3-carboxylic acid N -succinimidyl ester (4 mg) and diisopropylethylamine (6 µL) were successively added to a solution of LeuK0 (15 mg) in dimethylformamide (THF) (200 µL). These mixtures were stirred at room temperature at 800 rpm for 3 hours, and volatile constituents were evaporated at room temperature. Protein expression and purification For the expression of LcsG, E. coli ArcticExpress (DE3) carrying pACYC- lcsG was cultured. The E. coli cells were grown at 37°C in 1 L LB medium with appropriate antibiotics. IPTG (final concentration 0.1 mM) was supplemented to the culture when it reached an OD 600 at 0.6–0.8, and then the induced E. coli was grown at 11°C for 24 hours. The cells were harvested by centrifugation (5000 rpm, 15 min, 4°C), and resuspended in 20 mL lysis buffer (50 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl, 10 mM imidazole) and lysed by sonication on ice (200 W, 10 s, 10 s, 20 min) with an ultrasonic homogenizer SCIENTZ-IID (SCIENTZ, China). The lysate was centrifugated (12000 g, 1 hour, 4°C), then the supernatant was collected and filtered with a 0.45 µm membrane (Sartorius Stedim Biotech, Germany) to remove residual cellular debris. A nickel affinity chromatography column was prepared with 2 mL Ni-NTA Resin (Trans Gen Biotech, China) loaded into a gravity column (Sangon, China) and used for separation of the filtered supernatant. After washing with wash buffer (50 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl, 20/40/60 mM imidazole), His-tagged proteins were eluted with elution buffer (50 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl, 250 mM imidazole). Purified proteins were concentrated in the storage buffer (50 mM Tris-HCl, pH 7.5, 20% glycerol), and flash frozen. The purity of the protein was confirmed by SDS-PAGE. The mutant proteins were purified in a similar procedure with LcsG. Mutated proteins expressions were verified by Western blot using an anti-His antibody (TransGen Biotech Ltd, Beijing, China). In vitro enzyme assay for LcsG and its mutants To determine the function of LcsG, the enzyme assays (50µL) contained Tris-HCl buffer (50mM, pH 7.5), SAM (2 mM), LeuA or LeuK0 (10 µM), and the purified recombinant LcsG (10 µM). The reactions were incubated at 28°C for 120 min. For single-factor experiments, the enzyme assays (50µL) contained Tris-HCl buffer (50mM), SAM (2 mM), PLBJ-1 WT crude extract (200 µg), and the purified recombinant LcsG (10 µM). For the reaction time cause assay, LcsG was tested with Tris-HCl buffer (pH 7.5) at 28°C for 0.01 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min. For the reaction pH cause assay, LcsG was tested at 28°C for 120 min with Tris-HCl buffer in different pH (pH 7.0, pH 7.5, pH 8.0, pH 8.5). For the reaction temperature cause assay, LcsG was tested with Tris-HCl buffer (pH 8.0) for 120 min at 18°C, 21°C, 27°C, 34°C, and 38°C. To determine the function of mutated LcsG, 50 µL reaction systems were prepared with 500 nM catalysts, 2 mM SAM, 100 mM Tris-HCl buffer (pH 8.0), and 10 µM substrates. The reactions were incubated at 34°C for 120 min. The above enzymatic reactions were quenched by adding 50 µL cold acetonitrile and 10 µL was analyzed by LC-MS. Calibration curves of LeuA0 and LeuK3 Compounds were quantified by an external standard method and the construction of calibration curves using LeuA0 and LeuK3. In this study, LeuA0 was diluted at concentrations of 25, 50, 100, 250, and 500 nM. LeuK3 was also diluted to five concentrations of 50, 100, 250, 500, to 1000 nM. Michaelis-Menten enzyme kinetics Kinetic constants of LcsG were determined by the following approach: 1 µM LcsG was assayed against a range of substrate (LeuA or LeuK0) concentrations (2-200µM) with the initial rate of reaction measured by monitoring product formation (LeuA0 or LeuK3) at 34°C for 30 min. The rates were plotted against substrate concentration using the Michaelis-Menten kinetics equation by nonlinear regression analysis with the software GraphPad Prism 8 and K m and K cat constants generated from the resulting Michaelis-Menten plot. LC-MS analysis LC-MS analyses were run on an Agilent 1290 Infinity II HPLC with an Agilent Infinity Lab single quadrupole mass selective detector by using an Agilent Zorbax Eclipse Plus C18 reversed-phase column (2.1 × 100 mm, 2.7 µm). Water (A) with 0.1% (v/v) formic acid and acetonitrile (B) were used as the solvents at a flow rate of 0.25 mL min − 1 . The substances were eluted with 10% (v/v) B for 1 min, then a linear gradient from 10 to 100% (v/v) B in 12 min, washed with 100% solvent B for 5 min, and equilibrated with 5% solvent B for 10 min at a flow rate of 0.25 mL/min. The mass spectrometer was set in electrospray positive ion mode for ionization. LC-HRESI-MS-MS analyses were run on an Agilent HPLC 1260 Infinity II system equipped with an Agilent G6510A mass spectrometer by using an Agilent Zorbax SB-C18 reversed-phase column (4.6 × 150 mm, 5 µm). A linear gradient analytical method (10–100% MeCN in water with 0.1% formic acid for 20 min at a flow rate of 1.0 mL/min) was used. The Q-TOF was operated in positive mode with a capillary voltage of 1800 V and a drying gas flow rate of 1 µL/min at 300°C. MS scan range was 80–2000 m/z , and MS-MS scan range was 40–1400 m/z . Fixed collision energies were 65 V. Microbial growth inhibition assays Growth inhibition of leucinostatins (LeuK0, LeuK3, LeuA0) with C. neoformans H99 was assessed on PDA medium with agar diffusion assays. Overnight cultures of C. neoformans grown in PDB broth at 28°C were diluted with PDB broth to an OD 600 at 0.1. 1 mL aliquots of the resulting mixture were combined with 30 mL aliquots of PDA at 45°C. The test wells (4 mm diameter) were aspirated from the solidified medium using the tip of a sterilized Luer-lock syringe, and 10 µL of compounds were added to the wells. The plate was incubated at 28°C. Zones of inhibition were photographed after 36 hours. Leucinostatins (Solarbio, China) and Amphotericin B were dissolved in DMSO. Growth inhibition of leucinostatins (LeuK0, LeuK3, LeuA0) with P. infestans was assessed on rye agar medium in 9-cm Petri plates. P. infestans was incubated on the center of plates and cultured at 18°C for 3 d, followed by aspiration test wells (4 mm diam) at the colony edges. According to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations 50 , the MIC (minimal inhibitory concentration) was determined with three replicates using the serial dilution method in 96-well plates with YM (1% maltose extract, 0.2% yeast extract) as the test medium. Amphotericin B was used as the positive control. Test compounds were dissolved in DMSO and serially diluted in a growth medium. Visual endpoint and the optical density readings of microplate wells were measured relative to positive and negative controls. The strains were incubated at 25°C, and the MICs were determined at 48 hours for C. neoformans H99. Viability was determined with the aid of a plate reader using PrestoBlue resazurin dye (Life Technologies) as the viability indicator. The spectrophotometric MIC value was defined as the lowest concentration of a test compound that resulted in a culture with a density equal to 100% inhibition when compared to the growth of the untreated control. Structure prediction of LcsG Uni-fold was employed to predict LcsG dimer structure. A fasta file of two lcsG sequences was uploaded and a predicted structure was returned. The average pLDDT score was 0.88. Structure and sequence alignment Homology protein structures were searched via HHpred. ChimeraX was used to estimate the structural alignment and plot the figures. Multiple sequences were aligned by mafft, then espript3 was employed to export the sequence alignment results. Diffdock The structure of leucinostatin A was modified from the structure of its analogy ZHAWOC6027(PDB: 8a19). The Diffdock webserver was employed to simulate the dock. Predicted LcsG dimer structure and leucinostatin A structure were uploaded and the final docking poses were downloaded. The first rank docking pose was selected as the model. Declarations Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. Source data are provided with this paper. Acknowledgments This study was financially supported by the grants from the National Key R&D Program of China (2022YFD1400700), the National Natural Science Foundation of China (32272630) and the Agricultural Science and Technology Innovation Program of CAAS. We also thank Prof. Junfeng Liu and Dr. Xin Zhang (China Agricultural University, China) for their advice on protein purification. Author contributions Y, L., B. X, and Z. L. designed the research. Z. L. performed protein purification, fungal fermentation, compounds isolation, structure elucidation, LC-MS analysis, in vivo genetic and in vitro biochemical experiments; Y. J. performed protein purification, LC-MS analysis, in vivo genetic and in vitro biochemical experiments; J. L. and J. Z. performed the genomic analysis; Z. M. and Y. Y. assisted in the test of antimicrobial activity; K. Z. assisted in structure elucidation and chemical synthesis; Z.W. performed LC-MS analysis and LC-HRESI-MS-MS analysis; Y. L., B. X, and Z. L. wrote the manuscript. Ethics declarations Competing interests The authors declare no competing interests. References Schaner Tooley, C.E., et al.: NRMT is an α-N-methyltransferase that methylates RCC1 and retinoblastoma protein. Nature. 466 , 1125–1128 (2010) Stratton, C.F., Poulin, M.B., Du, Q., Schramm, V.L.: Kinetic isotope effects and transition state structure for human phenylethanolamine N -methyltransferase. ACS Chem. 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Prod. 79 , 2357–2363 (2016) Additional Declarations There is NO Competing Interest. Supplementary Files SI.docx nrreportingsummaryflat.pdf Reporting Summary Cite Share Download PDF Status: Published Journal Publication published 22 Jun, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3280468","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":233310968,"identity":"294cc338-5fa7-4ba5-b75f-0b331bb08997","order_by":0,"name":"Yan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYBACPmYQafBPjngtbGAtFQeMSdACJs8cSGwgXgs7j+HnwrY76f3thx9/YKi5Q4zDeIylZ7Y9y51xJs1MguHYM6K0GEjztjHnbmBIMGNgbDhMnC2/gVrSDfiff/5ArBYzaZ4zhxMMJHIMJIjUwlZmzVORZjjjxpsyiYRjRGjh5z+8+TaPgY08f3/65g8faojQwsDAYYBgJxCjgYGB/QFx6kbBKBgFo2DkAgCjnjN+osHbWgAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Li","suffix":""},{"id":233310969,"identity":"3eee2653-3f42-4809-a3f8-2f88dacfb475","order_by":1,"name":"Zixin Li","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zixin","middleName":"","lastName":"Li","suffix":""},{"id":233310970,"identity":"df86ade3-e84c-4328-96af-0b602d72e46b","order_by":2,"name":"Yang Jiao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Jiao","suffix":""},{"id":233310971,"identity":"a1daddb5-010c-40d1-ad06-0c840d43f9d6","order_by":3,"name":"Jian Ling","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Ling","suffix":""},{"id":233310972,"identity":"eb393829-c071-4c1c-837f-ea6ef81bb959","order_by":4,"name":"Jianlong zhao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianlong","middleName":"","lastName":"zhao","suffix":""},{"id":233310973,"identity":"9f6b90f2-74eb-49b6-8ac8-686c43ec57a0","order_by":5,"name":"Yuhong Yang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Yang","suffix":""},{"id":233310974,"identity":"9e4ee328-3f2d-449a-8e5e-c34898847955","order_by":6,"name":"Zhenchuan Mao","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenchuan","middleName":"","lastName":"Mao","suffix":""},{"id":233310975,"identity":"d4c20d14-62d8-482b-89d4-1cfb31e404d2","order_by":7,"name":"Kaixiang Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaixiang","middleName":"","lastName":"Zhou","suffix":""},{"id":233310976,"identity":"20f426d8-035d-43bd-af50-e024b4d73bb6","order_by":8,"name":"Wenzhao Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenzhao","middleName":"","lastName":"Wang","suffix":""},{"id":233310977,"identity":"4fe6df01-e9d7-4c3c-80c5-4a06e7e461ca","order_by":9,"name":"Bingyan Xie","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bingyan","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2023-08-20 20:40:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3280468/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3280468/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-024-06467-0","type":"published","date":"2024-06-22T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":43448538,"identity":"08712fab-fe95-401b-bed3-8ad4c3e4ec99","added_by":"auto","created_at":"2023-09-21 00:07:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of a predicted methyltransferase LcsG. (A) \u003c/strong\u003eStructures of leucinostatins (1-5). MeHA: methylhex-2-enoic acid; MePro: 4-methyl-proline; AHMOD: 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid; HyLeu: hydroxyleucine; Aib: aminoisobutyric acid; Leu: Leucine; \u003cem\u003eβ\u003c/em\u003eAla: \u003cem\u003eβ\u003c/em\u003e-Alanine.\u003cstrong\u003e (B) \u003c/strong\u003eGenetic organization of the leucinostatins BGC in \u003cem\u003eP. lilacinum\u003c/em\u003e PLBJ-1\u003cstrong\u003e. \u003c/strong\u003eMT: methyltransferase; PKS: polyketide synthase; NRPS: nonribosomal peptide synthetase.\u003cstrong\u003e (C) \u003c/strong\u003eLC-MS analysis of the lcsG knock-out (Δ\u003cem\u003elcsG\u003c/em\u003e) mutant and the wild-type strain (WT).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3280468/v1/5b7ee3674cd8af0c691f343f.png"},{"id":43448537,"identity":"54ccb930-a2c1-4294-ba0a-34f7a1984969","added_by":"auto","created_at":"2023-09-21 00:07:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVerification of LcsG involved in methylating leucinostatins. (A) \u003c/strong\u003eSDS-PAGE analysis of the recombinant protein LcsG. \u003cstrong\u003e(B) \u003c/strong\u003eIn vitro LcsG activity analysis using LeuK0 (\u003cstrong\u003e4\u003c/strong\u003e) and LeuA (\u003cstrong\u003e3\u003c/strong\u003e) as the substrate and the [M+H]\u003csup\u003e+\u003c/sup\u003e ions of three new product peaks \u003cstrong\u003e6\u003c/strong\u003e-\u003cstrong\u003e8\u003c/strong\u003e.\u003cstrong\u003e (C)\u003c/strong\u003e The time dependency of the variations of each component.\u003cstrong\u003e (D)\u003c/strong\u003e Kinetic analysis of lcsG using LeuA (\u003cstrong\u003e3\u003c/strong\u003e, up) and LeuK0\u0026nbsp; (\u003cstrong\u003e4\u003c/strong\u003e, down) as a substrate. The error bar indicates the standard deviation for triplicate measurements (n=3).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3280468/v1/598ccc56bf0368f138a1231c.png"},{"id":43448541,"identity":"51c3e7c5-5948-46d4-a537-7db2f9e5293e","added_by":"auto","created_at":"2023-09-21 00:07:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":762615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure elucidation of LeuA0 (8), LeuK2 (6), and LeuK3 (7). (A)\u003c/strong\u003e LC-MS analysis of \u003cem\u003eN\u003c/em\u003e-methylation of LeuA (\u003cstrong\u003e3\u003c/strong\u003e) to the same trimethylammonium compound (\u003cstrong\u003e8\u003c/strong\u003e, LeuA0) by LcsG and CH\u003csub\u003e3\u003c/sub\u003eI.\u003cstrong\u003e (B) \u003c/strong\u003eLC-MS analysis of two methylation reactions using LeuK0 (\u003cstrong\u003e4\u003c/strong\u003e) as substrate, and LcsG and CH\u003csub\u003e3\u003c/sub\u003eI. \u003cstrong\u003e(C) \u003c/strong\u003eStructures and the HERSI-MS-MS data of LeuA0 (\u003cstrong\u003e8\u003c/strong\u003e), LeuK0 (\u003cstrong\u003e4\u003c/strong\u003e), LeuK2 (\u003cstrong\u003e6\u003c/strong\u003e), and LeuK3\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003e7\u003c/strong\u003e). \u003cstrong\u003e(D)\u003c/strong\u003e Inhibition of growth of eukaryotic microorganisms by LeuK0 (\u003cstrong\u003e4\u003c/strong\u003e), LeuA0 (\u003cstrong\u003e8\u003c/strong\u003e), and LeuK3 (\u003cstrong\u003e7\u003c/strong\u003e) at 25 μg/well by agar diffusion assay.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3280468/v1/e736d8dc1063b57d06877107.png"},{"id":43448539,"identity":"572b98be-ef1a-4065-b311-3ef044e70724","added_by":"auto","created_at":"2023-09-21 00:07:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":376738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatalytic sites of LcsG. (A) \u003c/strong\u003eSAH binding sites of OxaC (PDB code: 5w7p, marked in pink), LepI (PDB code: 6ix7, marked in blue), and LcsG (marked in golden).\u003cstrong\u003e (B) \u003c/strong\u003eModified LeuA binding sites of LcsG. \u003cstrong\u003e(C) \u003c/strong\u003eComparison of wild-type LcsG and mutated LcsG-mediated methylations. The yield of the product was quantified by comparison with the peak area of those catalyzed by LcsG (100%).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3280468/v1/6f3e748b058ed6d1e1ca796e.png"},{"id":58881051,"identity":"4f63dde5-cfb2-456e-8fe4-05e628d11fcc","added_by":"auto","created_at":"2024-06-23 07:06:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2238644,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3280468/v1/79b139a6-7f88-4cda-afce-b03489a3387e.pdf"},{"id":43448543,"identity":"1d48adb8-5607-4f7c-b1dd-e4492473b96d","added_by":"auto","created_at":"2023-09-21 00:07:49","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3471619,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-3280468/v1/1c54571efd7cac3d32ed0e41.docx"},{"id":43448540,"identity":"5d963b0f-301f-453a-977a-fd4d4e41a51b","added_by":"auto","created_at":"2023-09-21 00:07:49","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":781519,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"nrreportingsummaryflat.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3280468/v1/1106b964ebce451822360bdb.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eN\u003c/em\u003e-methyltransferases (NMTs) are ubiquitous tailoring enzymes found in all kingdoms of life. NMTs often function as the modulator of signaling molecules in animal signal transduction pathways.\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e NMTs are also employed in the biosynthetic pathways of natural products.\u003csup\u003e4\u0026ndash;7\u003c/sup\u003e The \u003cem\u003eN\u003c/em\u003e-methylations catalyzed by NMTs contribute significantly to changing the property of natural products by modifying their structures and influencing their bioactivities.\u003csup\u003e8\u003c/sup\u003e In the biosynthesis of nonribosomal peptides (NRPs), NMTs often integrate into the nonribosomal peptides synthase (NRPS) as domains, while seldom acting as a freestanding enzyme.\u003csup\u003e9,10\u003c/sup\u003e Although the existing examples describe enzymatic mono-\u003cem\u003eN\u003c/em\u003e-methylations in peptide bonds or side chains of NRPs,\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e NMT-catalyzed iterative \u003cem\u003eN\u003c/em\u003e-methylation at the terminus of NRPs has been rarely reported.\u003c/p\u003e \u003cp\u003eLeucinostatins are a family of lipopeptide antibiotics, derived from \u003cem\u003ePurpureocillium lilacinum\u003c/em\u003e.\u003csup\u003e14\u003c/sup\u003e They exhibit a wide range of biological activities affecting multiple pathogens.\u003csup\u003e15\u003c/sup\u003e Furthermore, they have been studied as potential anticancer agents and potent antiprotozoal agents.\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e Their bioactivities are attributed to the inhibition of mitochondrial function.\u003csup\u003e19,20\u003c/sup\u003e At least 24 leucinostatin homologues have been isolated and characterized.\u003csup\u003e21\u003c/sup\u003e Their backbone chains are assembled by a NRPS with nine amino acids and an atypical amino moiety at \u003cem\u003eC\u003c/em\u003e-terminus, linked by peptide linkages.\u003csup\u003e22\u003c/sup\u003e The \u003cem\u003eC\u003c/em\u003e-terminuses of leucinostatins are methylated to varying degrees. Specifically, the \u003cem\u003eC\u003c/em\u003e-terminus of leucinostatin C (\u003cb\u003e1\u003c/b\u003e, LeuC) is propane-1,2-diamine (PD), while that of leucinostatin B (\u003cb\u003e2\u003c/b\u003e, LeuB) and leucinostatin A (\u003cb\u003e3\u003c/b\u003e, LeuA) are protected by \u003cem\u003eN\u003c/em\u003e-methylpropane-1,2-diamine (MPD), and \u003cem\u003eN\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e-dimethylpropane-1,2-diamine (DMPD), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). However, the biosynthetic mechanisms of the diverse \u003cem\u003eC\u003c/em\u003e-terminuses remain unknown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this work, we identify a discrete NMT that could catalyze a unique moiety located in the terminus of a NRP, named as LcsG, from the biological control fungus \u003cem\u003eP. lilacinum\u003c/em\u003e PLBJ-1.\u003csup\u003e23\u003c/sup\u003e Deleting the \u003cem\u003elcsG\u003c/em\u003e gene led to the disappearance of leucinostatins with methylated terminus. Subsequent in vitro enzyme activity assays and structure elucidations of products demonstrate the LcsG conducts iterative methylation at the terminal-free amines of leucinostatins. Moreover, the structure-function relationship analysis provided a probable insight into the catalytic mechanism of LcsG. Additionally, we obtained new leucinostatins from the LcsG-catalyzed reaction, which could inhibit the growth of the human pathogen \u003cem\u003eCryptococcus neoformans\u003c/em\u003e and the plant pathogen \u003cem\u003ePhytophthora infestans\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of LcsG from\u003c/b\u003e \u003cb\u003ePurpureocillium lilacinum\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous studies demonstrated that the strain \u003cem\u003eP. lilacinum\u003c/em\u003e PLBJ-1 harbors the leucinostatin biosynthetic gene cluster (BGC),\u003csup\u003e23\u003c/sup\u003e and the gene \u003cem\u003elcsG\u003c/em\u003e whose function was predicted as a methyltransferase attracted our interest (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The LcsG contained the Methyltransf_2 domain that is typical for \u003cem\u003eO\u003c/em\u003e-methyltransferases (OMTs) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and the top hit (31.65% sequence identity) of its pBLAST research was an OMT, VdtC (A0A443HJY8.1).\u003csup\u003e24\u003c/sup\u003e Despite this prediction, we could not find an \u003cem\u003eO\u003c/em\u003e-methylated unit in leucinostatins. This discrepancy prompted our curiosity to analyze the function of LcsG.\u003c/p\u003e \u003cp\u003eTo figure out the function of LcsG in the leucinostatins biosynthesis, we constructed a deletion mutant (Δ\u003cem\u003elcsG\u003c/em\u003e) and an overexpression mutant (OE\u003cem\u003elcsG\u003c/em\u003e) of \u003cem\u003eP. lilacinum\u003c/em\u003e PLBJ-1 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3). Following growth on a productive medium and production extraction, LC-MS analysis suggested obvious differences between the deletion mutant and wild-type strain (WT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), while there were no obvious differences between the overexpression mutant and WT (Fig. S3). The LC-MS analysis of WT extracts showed the \u003cem\u003em/z\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e of four peaks (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) to be 1190.8133, 1218.8439, 1204.8247, and 1234.8388, respectively (Fig. S4). High-resolution electrospray ionization mass spectrometry (HRESI-MS-MS) analysis (Table S2 and Fig. S5) confirmed peaks \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e3\u003c/b\u003e as the known LeuC, LeuB, and LeuA, respectively. HRESI-MS-MS and NMR analysis suggested peak \u003cb\u003e4\u003c/b\u003e was not the known compound leucinostatin K (LeuK), but a compound derived from LeuC whose \u003cem\u003eC\u003c/em\u003e-terminus was NH-CH\u003csub\u003e2\u003c/sub\u003e-CH\u003csub\u003e2\u003c/sub\u003e-OH or CH\u003csub\u003e2\u003c/sub\u003e-CH\u003csub\u003e2\u003c/sub\u003e-O-NH\u003csub\u003e2\u003c/sub\u003e and designated as leucinostatin K0 (\u003cb\u003e4\u003c/b\u003e, LeuK0) (Fig. S6-S8). A specific \u003cem\u003eN\u003c/em\u003e-hydroxysuccinimide (NHS)-ester reaction was employed to verify the free amine in LeuK0, and then the product compound LeuK1 (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) with NHS ester labeling was detected (Fig. S9), which confirmed the structure of LueK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The deletion of \u003cem\u003elcsG\u003c/em\u003e only led to the abolishment of LeuB and LeuA, suggesting the deletion blocked the formation of the methylated \u003cem\u003eC\u003c/em\u003e-terminal amines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Therefore, LcsG was inferred to play an essential role in the biosynthesis of the terminal amines of leucinostatins.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLcsG functions as a SAM-dependent methyltransferase\u003c/h2\u003e \u003cp\u003eSequence analysis suggested that LcsG is an \u003cem\u003eS\u003c/em\u003e-adenosyl-\u003csub\u003eL\u003c/sub\u003e-methionine (SAM)-dependent methyltransferase. To clarify its biochemical function, we expressed \u003cem\u003elcsG\u003c/em\u003e in \u003cem\u003eE. coli\u003c/em\u003e ArcticExpress (DE3) and purified the recombinant His\u003csub\u003e6\u003c/sub\u003e-tagged protein LcsG by nickel affinity chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The enzymatic activity was assayed using the LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) as the substrates, respectively. The S-adenosyl-\u003csub\u003eL\u003c/sub\u003e-homocysteine (SAH) and three new product peaks LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) were only detected in the presence of the substrate, LcsG, and SAM (Fig. S10 and 2B). In contrast, omitting either LcsG or SAM resulted in no product formation. LC-MS analysis proved the [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e of ions of \u003cb\u003e6\u003c/b\u003e\u0026ndash;\u003cb\u003e8\u003c/b\u003e were 1248.8533, 1262.8711, and 1232.8595, respectively (Fig. S11). These molecular weights indicated LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) should be the methylated products of LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), respectively, and LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) should be the dimethylated product of LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These results indicated that LcsG is likely a SAM-dependent methyltransferase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe kinetic activity of LcsG was analyzed to gain further insight into the methylation activity. We conducted several single-factor enzymatic assays by using the crude extract of WT as the substrate and compared the differences between the control groups and the experimental groups by LC-MS to measure the variations of each component. The time dependency of the LcsG-catalyzed reaction showed that LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) accumulated nearly linearly with time and appeared almost immediately after the reaction started. LeuB (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) and LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) were nearly completely consumed after the beginning of the reaction, while the amount of LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) increased early on, then decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results suggested that LcsG is an iterative methyltransferase, and the reaction sequences could be LeuB-LeuA-LeuA0 and LeuK0-LeuK2-LeuK3. The optimal pH and temperature for producing the final products LeuA0 and LeuK3 were determined (Fig. S12), followed by the measurements of the initial rates at substrate concentrations ranging from 0-200 \u0026micro;M. The initial rate data were measured by LC-MS and fitted to the Michaelis-Menten equation to derive kinetic parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Fig. S13). The \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values were 65.39 s\u003csup\u003e-1\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e and 32.94 s\u003csup\u003e-1\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e towards LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), respectivelyand LeuA, respectively, and LeuK3 might be the dimethylated product of LeuK0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCharacterizations of the products of LcsG-catalyzed reaction\u003c/h2\u003e \u003cp\u003eTo elucidate the structures of compounds LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), we turned to HRESI-MS-MS analysis. The \u003cem\u003em/z\u003c/em\u003e values of fragments of each leucinostatins were presented in Table S2. Comparisons of the MS-MS data of LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) with those data of LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and LeuB (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), found that the spectrum of LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) showed remarkably similar fragments with LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and LeuB (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) (Fig. S14). Specifically, they shared one same fragment whose \u003cem\u003em/z\u003c/em\u003e value was 1173. This ion was deduced to be the [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e ion of the fragment C10, indicating the possible methylated site of the \u003cem\u003eC\u003c/em\u003e-terminal amine. Therefore, LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) was concluded to be a trimethylammonium compound in which the terminal amine carried a positive charge and three methyl groups. This predicted structure is identical to a previously identified structure,\u003csup\u003e25\u003c/sup\u003e which was obtained by treating LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) with methyl iodide. This reaction was prepared and gave the same product as the enzymatic reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), confirming the structures of LeuA0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Combined with the molecular weight, LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) was assigned the molecular formula C\u003csub\u003e63\u003c/sub\u003eH\u003csub\u003e114\u003c/sub\u003eN\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, treating LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) with methyl iodide also gave the products LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). According to the molecular weights of LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), their molecular formulas should be C\u003csub\u003e63\u003c/sub\u003eH\u003csub\u003e113\u003c/sub\u003eN\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e and C\u003csub\u003e64\u003c/sub\u003eH\u003csub\u003e115\u003c/sub\u003eN\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e, respectively. From the MS-MS experiment, they also presented similar fragments as LeuK0 (Fig. S15). The differences were observed in the fragments belonging to the Y type. The Y type fragments in LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) are +\u0026thinsp;14 mass units greater than the corresponding fragments observed in LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). For example, the \u003cem\u003em/z\u003c/em\u003e values 190 and 204 refer to [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e ions of fragments Y2 in LeuK0 and LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), respectively. Furthermore, LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) also afforded an ion at \u003cem\u003em/z\u003c/em\u003e 1173 (C10) indicating the possible methylation of the terminal amine of LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Additionally, LeuK2 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) were new leucinostatins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results demonstrated that the LcsG is a NMT that could iteratively catalyze the methylation of the terminal amine of NRP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAntifungal evaluation of leucinostatins\u003c/h2\u003e \u003cp\u003eLeucinostatins are well-known antibiotics. We carried out the purification for the compounds and obtained LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), LeuK0 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) and LeuK3 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) in sufficient quantities to do the antifungal assay. We determined the inhibitory activity of these compounds against the drug resistant strain \u003cem\u003eC. neoformans\u003c/em\u003e H99 and the plant pathogen \u003cem\u003eP. infestans\u003c/em\u003e by agar diffusion assays. All these leucinostatins showed inhibitory effects against these pathogens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Moreover, the anti-\u003cem\u003eC. neoformans\u003c/em\u003e MIC value of the two methylated products, LeuA0 (\u003cb\u003e8\u003c/b\u003e, 25.8 \u0026micro;g/mL) and LeuK3 (\u003cb\u003e7\u003c/b\u003e, 25.8 \u0026micro;g/mL), were four and two times lower than those of their parent molecules, LeuA (\u003cb\u003e3\u003c/b\u003e, 102.4 \u0026micro;g/mL) and LeuK0 (\u003cb\u003e4\u003c/b\u003e, 51.2 \u0026micro;g/mL), respectively (Fig. S16), which indicated that \u003cem\u003eN\u003c/em\u003e-methylation at the terminus of leucinostatins could improve their antifungal efficiencies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCatalytic mechanism of\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-methyltransferase LcsG\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe then identified the catalytic residues in LcsG. Local multiple sequence alignments revealed that LcsG shared a conserved SAH/SAM binding motif. Despite many attempts, we were unable to obtain a crystal of LcsG protein suitable for X-ray crystallographic analysis. As an alternative, we employ artificial intelligence (AI) methods to approximate a model of LcsG. The overall structure of LcsG is a typical ClassI methyltransferase fold, the \u003cem\u003eN\u003c/em\u003e-terminus appears responsible for dimerization and substrate binding and the \u003cem\u003eC\u003c/em\u003e-terminus appears responsible for SAM binding. To unravel the structure-function relationship of LcsG, we conducted a molecular docking analysis using the predicted LcsG structure and SAH. SAH was docked into the LcsG structure model\u0026rsquo;s binding pocket and the hydrogen bonds network that mediates SAH binding was present in\u003c/p\u003e \u003cp\u003ethe final docking position. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, potential hydrogen bond interactions between SAH and residues Asp296, Asp321, Asp348, and Lys363 were presented, which was consistent with the result of multiple sequence alignment (Fig. S17). The accurate stereo-structure structures of nonpeptide leucinostatins are hard to predict because they comprised seven non-standard and unusual α-amino acid residues. Based on the crystal structures of analogues helioferin A\u003csup\u003e26\u003c/sup\u003e and ZHAWOC6027\u003csup\u003e18\u003c/sup\u003e, structural model of LeuA was predicted, and docked into LcsG structure via Diffdock.\u003csup\u003e27\u003c/sup\u003e In the first ranked result, the \u003cem\u003eN\u003c/em\u003e atom which would be methylated occupies the position between Asp368, Asp395 and SAH (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify this, we mutated Asp296, Asp321, Asp348, Lys363, Asp368, and Asp395 to Ala in LcsG (Fig. S18). Biochemical assays of these mutants were then performed using LeuA as substrate. After 1 hour incubation, all mutants showed decreases in the conversion of LeuA (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) to LeuA0 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results were aligned with the previous results of in vitro assays and molecular docking, suggesting the binding site of SAM of LcsG in leucinostatins methylation and further supported that LcsG was a SAM-dependent methyltransferase.\u003c/p\u003e \u003cp\u003eThe reaction mechanism of OMT has been proposed in several studies.\u003csup\u003e28\u003c/sup\u003e In the proposed mechanism, a His/Glu dyad and an Asp residue were required. The Glu residue was placed near the His residue and activated the His residue to deprotonate the hydroxyl group in the substrate. The Asp residue was shown to interact with the substrate for improved binding. The deprotonated hydroxyl group would function as a good nucleophile to attack the SAM, which is the methyl donor, to form the \u003cem\u003eO\u003c/em\u003e-methylation product. For NMT, similar but different mechanisms have been proposed.\u003csup\u003e29\u003c/sup\u003e A QM/MM study on the catalytic mechanism of phenylethanolamine NMT\u003csup\u003e30\u003c/sup\u003e stated that unlike OMT, a Glu residue was employed in the deprotonation step. The protonated amine in the substrate was deprotonated by a Glu residue to form a nucleophile. Then, the methyl group was transferred from the methyl-donor SAM to the deprotonated amine group.\u003c/p\u003e \u003cp\u003eFor LcsG, the mutation data and docking result indicated that the reaction mechanism of LcsG is like phenylethanolamine NMT. Unlike reactions catalyzed by OMT, a His/Glu dyad was not required. The lone electron pair of the \u003cem\u003eN\u003c/em\u003e atom on the dimethylamine group could undergo a nucleophilic attack. This was consistent with the fact that in sequence alignment, the corresponding residue of OMT His was His367 (Fig. S19). We proposed that the protonated dimethylamine group in the leucinostatins is coordinated and deprotonated by two negatively charged residues (D368, D395). A nucleophilic attack between the dimethylamine and SAM follows and the methyl group was transferred from SAM to leucinostatins. Compared to wild-type LcsG, the LcsG-D368A and LcsG-D395A showed obvious decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating these two residues contribute significantly to the substrate binding.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNMTs are important for the biotransformation of bioactive molecules. The \u003cem\u003eN\u003c/em\u003e-methylation can modulate the activity of signaling molecules and participate in the biosynthesis of natural products. NMTs are of great interest because the site-specific modification is crucial for the bioactivity and biosynthesis of natural products. In this study, we successfully determined the in vitro activity of the NMT, LcsG, involved in the iterative \u003cem\u003eN\u003c/em\u003e-methylation of the unique terminal unit of leucinostatins. Few NMTs are known to iteratively transfer methyl groups to natural products. The mycobacterial NMT EgtD\u003csup\u003e31\u003c/sup\u003e, \u003cem\u003ePlasmodium falciparum\u003c/em\u003e PfPMT\u003csup\u003e32\u003c/sup\u003e, and human NRMT (\u003cem\u003eN\u003c/em\u003e-terminal RCC1 methyltransferase)\u003csup\u003e1\u003c/sup\u003e were reported to progressively catalyze \u003cem\u003eN\u003c/em\u003e-trimethylation of corresponding substrates. The \u003cem\u003ePsilocybe\u003c/em\u003e NMT PsiM\u003csup\u003e33\u003c/sup\u003e was shown the capacity of catalyzing geminal \u003cem\u003eN\u003c/em\u003e-dimethylation. Although the NMTs involved in the biosynthesis of plantazolicin class ribosomally synthesized and posttranslationally modified peptides (RiPPs) are responsible for the dimethylation, the corresponding monomethylated products were never detected\u003csup\u003e8,34\u003c/sup\u003e. Besides, the phylogenetic analysis of LcsG and all related NMTs suggested that LcsG is closely related to OMTs although it clusters with NMT (Fig. S20).\u003c/p\u003e \u003cp\u003eNRPSs are well-known megaenzymes that consist of sequential domains. The peptide is elongated followed by released from the terminal module, including condensation domains (C\u003csub\u003eT\u003c/sub\u003e), reductase domains (R), Dieckmann cyclase domains (D), and thioesterase domains (TE)\u003csup\u003e35\u003c/sup\u003e. According to the analysis of antiSMASH 2.0 and pBLAST results (Table S3), the terminal module of the NRPS in the biosynthetic gene cluster (LcsA) should be an R domain that could release the peptide from the NRPS by hydrolysis, so that the \u003cem\u003eC\u003c/em\u003e-terminus of leucinostatins normally should be an aldehyde group\u003csup\u003e36\u0026ndash;39\u003c/sup\u003e or a hydroxyl group\u003csup\u003e40\u0026ndash;42\u003c/sup\u003e (Fig. S21). However, we have not seen such similar structures among the existing characterized leucinostatins. Based on these reports and the NMR results of LeuK0, we initially deduced the \u003cem\u003eC\u003c/em\u003e-terminal of LeuK0 to be NH-CH\u003csub\u003e2\u003c/sub\u003e-CH\u003csub\u003e2\u003c/sub\u003e-OH, but this hypothesis was rejected by the subsequent NHS ester reaction and enzyme assays.\u003c/p\u003e \u003cp\u003eFor OMT OxaC and CHOMT, the key catalytic sites responsible for catalyzing the methyltransfer reaction were reported as His313 and His278, respectively. Interestingly, the corresponding residue in LcsG is Tyr according to the multiple sequence alignments. The multiple sequence alignment result suggested the corresponding residues in other reported OMTs were basic residues (His), and residues in NMTs were neutral or acidic (Fig. S22). The LcsG-catalyzed \u003cem\u003eN\u003c/em\u003e-methyl transfer reaction is expected to occur via nucleophilic attack by the lone electron pair of terminal \u003cem\u003eN\u003c/em\u003e of leucinostatins on the reactive sulfonium methyl group of SAM\u003csup\u003e43\u003c/sup\u003e. The \u003cem\u003eO\u003c/em\u003e-methyltransfer reaction needs a base-assisted deprotonation step to generate a nucleophile\u003csup\u003e44\u003c/sup\u003e, and this may be the reason why OMTs share a His residue. On the contrary, the deprotonation of the \u003cem\u003eN\u003c/em\u003e-methyltransfer reaction does not require a basic residue.\u003c/p\u003e \u003cp\u003eIn conclusion, an OMT-like enzyme from \u003cem\u003eP. lilacinum\u003c/em\u003e, LcsG, was identified as a discrete SAM-dependent NMT which can iteratively catalyze the primary amine, secondary amine, and tertiary amine in the unique terminal unit of leucinostatins. Furthermore, one new secondary metabolite (LeuK0) and two enzymatic products (LeuK2 and LeuK3) were identified as new leucinostatins. In addition, the methylated compounds were observed to display higher antifungal activities than their parent molecules. To our knowledge, LcsG is a rare NMT that can methylate the terminal residues of NRPs. We expect that the results of this study provide deeper insights into mechanisms of \u003cem\u003eN\u003c/em\u003e-methylation of peptides and create the possibility of engineering new methylated molecules for exploring more potent antibiotics.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStrains and cultural conditions\u003c/h2\u003e \u003cp\u003eThe strains used in this study are listed in Supplementary Table\u0026nbsp;4. The \u003cem\u003ePurpureocillium lilacinum\u003c/em\u003e strain PLBJ-1 (CGMCC3.17492)\u003csup\u003e23,45,46\u003c/sup\u003e and its transformants were cultured at 28\u0026deg;C in potato dextrose agar (PDA) or potato dextrose broth (PDB) with appropriate antibiotics as required. \u003cem\u003eEscherichia coli\u003c/em\u003e Trelief 5α (Tsingke, China) was cultured at 37\u0026deg;C in Luria-Bertani (LB) broth with appropriate antibiotics. The \u003cem\u003eE. coli\u003c/em\u003e ArcticExpress (DE3) (Agilent Technologies) was used for LcsG protein expression. \u003cem\u003eE. coli\u003c/em\u003e ArcticExpress (DE3) was cultivated at 37\u0026deg;C in LB broth with appropriate antibiotics for growth, followed by growth at 11\u0026deg;C for inducing the recombinant protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis of LcsG and phylogenetic tree construction\u003c/h2\u003e \u003cp\u003eSequences of MTs were downloaded from the NCBI database. Multiple sequences were aligned with MUSCLE. The phylogenetic trees were constructed using the neighbor-joining method with bootstrap support of 1000 in MEGAX. The trees were visualized by using the Interactive Tree of Life (ITOL, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://itol.embl.de/\u003c/span\u003e\u003cspan address=\"http://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The amino acid sequence of LcsG was used as a query for BLASTp analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDNA and RNA isolation\u003c/h2\u003e \u003cp\u003eThe mycelia of PLBJ-1 and mutants were harvested via filtration. The genomic DNA was extracted using a Qiagen DNeasy Kit. The RNA was extracted using a TRIZOL reagent (Takara, Japan) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGene cloning and plasmid construction\u003c/h2\u003e \u003cp\u003eThe oligonucleotide sequences for PCR primers are listed in Supplementary Table\u0026nbsp;5. PCR reactions were performed using 2 \u0026times; Phanta Max Master Mix Polymerase (P525, Vazyme Biotech Co., Ltd, China) and Q5 High-Fidelity DNA Polymerase (New England Biolabs, USA).\u003c/p\u003e \u003cp\u003eThe plasmids are listed in Supplementary Table\u0026nbsp;4. To construct the deletion cassette of \u003cem\u003elcsG\u003c/em\u003e, about 1 kb DNA fragments located upstream and downstream of the \u003cem\u003elcsG\u003c/em\u003e coding region were amplified from the gDNA of PLBJ-1, named \u003cem\u003elcsGup\u003c/em\u003e and \u003cem\u003elcsGdown\u003c/em\u003e, respectively. Two fragments and the selection marker gene \u003cem\u003eneo\u003c/em\u003e were integrated into the \u003cem\u003eKpn\u003c/em\u003eI/\u003cem\u003eBam\u003c/em\u003eHI-cleaved vector pKOV21 via the digestion-ligation method by using T4 DNA Ligase (Thermo Fisher Scientific, USA) to give the deletion plasmid pKOV21-ko\u003cem\u003elcsG\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFor the overexpression of \u003cem\u003elcsG\u003c/em\u003e in PLBJ-1, the \u003cem\u003elcsG\u003c/em\u003e gene was amplified from the cDNA of PLBJ-1. The selection marker gene \u003cem\u003eneo\u003c/em\u003e and the terminator \u003cem\u003eTrpC\u003c/em\u003e were amplified from the KSTNP vector. This fragment and two restriction enzyme cutting sites \u003cem\u003ePme\u003c/em\u003eI and \u003cem\u003ePac\u003c/em\u003eI were integrated into the pEASY vector by using \u003cem\u003epEASY\u003c/em\u003e\u0026reg;-Blunt cloning Kit (TransGen Biotech, China) to obtain the intermediate vector pEASY-\u003cem\u003eneoTrpC\u003c/em\u003e. Then the strong promoter \u003cem\u003egpdA\u003c/em\u003e was amplified from the PCH-sGFP vector and integrated into the \u003cem\u003eNot\u003c/em\u003eI/\u003cem\u003eApa\u003c/em\u003eI-cleaved vector pEASY-\u003cem\u003eneoTrpC\u003c/em\u003e via the digestion-ligation method by using DNA T4 Ligase to obtain the vector pGNT. Afterwards, the \u003cem\u003elcsG\u003c/em\u003e gene was integrated into the \u003cem\u003ePme\u003c/em\u003eI/\u003cem\u003eNot\u003c/em\u003eI-cleaved vector pGNT by using \u003cem\u003epEASY\u003c/em\u003e\u0026reg;-Basic Seamless Cloning and Assembly Kit (TransGen Biotech, China) to obtain the overexpression vector pGNT-\u003cem\u003elcsG\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe recombinant protein LcsG expression vector pACYC-\u003cem\u003elcsG\u003c/em\u003e was generated by integrating the \u003cem\u003elcsG\u003c/em\u003e gene from the PLBJ-1 cDNA into the protein expression vector pACYCDuet-1 by using the Quick-change method\u003csup\u003e47\u003c/sup\u003e. The mutated LcsG protein vectors were obtained by using QuickMutation\u0026trade; Site-Directed Mutagenesis Kit (D02065, Beyotime Biotechnology, China) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePEG-mediated fungal transformation\u003c/h2\u003e \u003cp\u003eThe split-marker strategy was used in disrupting the \u003cem\u003elcsG\u003c/em\u003e gene. The DNA fragments \u003cem\u003elcsGup\u003c/em\u003e-\u003cem\u003ene\u003c/em\u003e (\u003cem\u003elcsGup\u003c/em\u003e and the first half of \u003cem\u003eneo\u003c/em\u003e) and \u003cem\u003eeo\u003c/em\u003e-\u003cem\u003elcsGdown\u003c/em\u003e (the second half of \u003cem\u003eneo\u003c/em\u003e and \u003cem\u003elcsGdown\u003c/em\u003e) were amplified from pKOV21-ko\u003cem\u003elcsG\u003c/em\u003e. The two fragments of \u003cem\u003eneo\u003c/em\u003e overlapped by 667 bp. 5 \u0026micro;g of two DNA fragments were transformed into PLBJ-1 by the polyethylene glycol (PEG)-mediated fusion of protoplasts according to the described protocol\u003csup\u003e45\u003c/sup\u003e. Geneticin (G418) resistant colonies were selected after culturing on PDA at 28\u0026deg;C for 1 day. The candidate transformants were picked and inoculated into new PDA plates with 400 \u0026micro;g/mL G418 (Inalco, USA) (400 \u0026micro;g/mL). These transformants were verified via diagnostic PCR with primers.\u003c/p\u003e \u003cp\u003eFor the overexpression of \u003cem\u003elcsG\u003c/em\u003e, the plasmid pGNT-\u003cem\u003elcsG\u003c/em\u003e and the empty vector pGNT were transformed into PLBJ-1 to construct the overexpression and control strain, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR analysis of\u003c/b\u003e \u003cb\u003elcsG\u003c/b\u003e \u003cb\u003eoverexpression strain\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor cDNAs synthesis, about 1 \u0026micro;g of DNase-treated, RNase-cleaned RNA was used as the template by using the HiScript III RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (R312, Vazyme Biotech Co., Ltd, China). Three biological replicates were measured for each analysis of the relative expression levels. The housekeeping gene \u003cem\u003eactin\u003c/em\u003e (Genebank number VFPBJ_07912) was used as a control. The qRT-PCR was run with ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech Co., Ltd, China) on a BIO-RAD CFX96 (BIO-RAD). The relative expression values were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method\u003csup\u003e48\u003c/sup\u003e. Primers are listed in Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCulture extraction\u003c/h2\u003e \u003cp\u003ePLBJ-1 and its mutants were cultured in PDB medium at 28\u0026deg;C and 220 rpm for 14 days. The fermentation was extracted with an equal volume of ethyl acetate (EtOAc) three times (each 1 hour) and EtOAc evaporated under reduced pressure. The extract was redissolved in acetonitrile (MeCN) for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChemical methylation of LeuA and LeuK0\u003c/h2\u003e \u003cp\u003eFor chemical methylation of LeuA and LeuK0, diisopropylethylamine (15 \u0026micro;L) and iodomethane (55 \u0026micro;L) were successively added to a solution of LeuA and LeuK0 (10 mg) in dry tetrahydrofuran (THF) (0.6 mL), respectively. These mixtures were stirred at room temperature at 800 rpm for 46 hours, and volatile constituents were evaporated at room temperature\u003csup\u003e49\u003c/sup\u003e. For isolating LeuA0 and LeuK3, the WT and Δ\u003cem\u003elcsG\u003c/em\u003e mutant crude extract were used as substrates, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProduct purification of LeuK0, LeuA0, and LeuK3\u003c/h2\u003e \u003cp\u003eTo isolate LeuK0, the Δ\u003cem\u003elcsG\u003c/em\u003e mutant was inoculated into PDB medium and incubated at 28\u0026deg;C for 14 days. LeuA0 and LeuK3 were isolated from chemically methylated WT and Δ\u003cem\u003elcsG\u003c/em\u003e mutant crude extract, respectively. LeuK0, LeuA0, and LeuK3 were purified by semi-preparative HPLC from crude extracts mentioned above, respectively. The UV absorption of leucinostatins was monitored at 214 nm with the HPLC\u0026rsquo;s DAD. Samples were separated on an Agilent 1260 Infinity II HPLC system with a Kromasil 100-5-C18 column (10 mm \u0026times; 250 mm), eluted with a linear gradient of 20\u0026ndash;70% of MeCN-water for 25 min at a flow rate of 2 mL/min. The retention time of LeuK0, LeuA0, and LeuK3 was 23.8 min, 21.6 min, and 22.3 min, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStructure characterization of LeuK0\u003c/h2\u003e \u003cp\u003eThe compound LeuK0 was assigned a molecular formula of C\u003csub\u003e62\u003c/sub\u003eH\u003csub\u003e111\u003c/sub\u003eN\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e on the base of HRESIMS (\u003cem\u003em/z\u003c/em\u003e 1234.8383 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e). Its ESI-MS-MS data were compared with those of previously reported leucinostatins A-C (Table S3). The spectra of these compounds showed the same fragments (from B1 to C10), which indicated that their structural feature (from B1 to C10) is the same and the \u003cem\u003eC\u003c/em\u003e-terminal unit (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eNO) in LeuK0 is different. Analysis of its \u003csup\u003e13\u003c/sup\u003eC-NMR APT, DEPT-135, and DEPT-90 spectroscopic data (Fig. S7-S9) revealed a total of 62 carbons, including 18 methyl groups (-CH\u003csub\u003e3\u003c/sub\u003e), 14 methylenes (-CH\u003csub\u003e2\u003c/sub\u003e), 16 methines (-CH), and 14 sp\u003csup\u003e3\u003c/sup\u003e quaternary carbons. Since the same unit (from B1 to C10) already contains 18 -CH\u003csub\u003e3\u003c/sub\u003e, 12 -CH\u003csub\u003e2\u003c/sub\u003e, 16 -CH, and 14 sp\u003csup\u003e3\u003c/sup\u003e quaternary carbons, thus the \u003cem\u003eC\u003c/em\u003e-terminal unit (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eNO) in LeuK0 was thought to be -CH\u003csub\u003e2\u003c/sub\u003e-CH\u003csub\u003e2\u003c/sub\u003e-O-NH\u003csub\u003e2\u003c/sub\u003e or CH\u003csub\u003e2\u003c/sub\u003e-CH\u003csub\u003e2\u003c/sub\u003e-OH.\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-hydroxysuccinimide (NHS)-ester reaction\u003c/b\u003e \u003c/p\u003e \u003cp\u003e7-Methoxycoumarin-3-carboxylic acid \u003cem\u003eN\u003c/em\u003e-succinimidyl ester (4 mg) and diisopropylethylamine (6 \u0026micro;L) were successively added to a solution of LeuK0 (15 mg) in dimethylformamide (THF) (200 \u0026micro;L). These mixtures were stirred at room temperature at 800 rpm for 3 hours, and volatile constituents were evaporated at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eFor the expression of LcsG, \u003cem\u003eE. coli\u003c/em\u003e ArcticExpress (DE3) carrying pACYC-\u003cem\u003elcsG\u003c/em\u003e was cultured. The \u003cem\u003eE. coli\u003c/em\u003e cells were grown at 37\u0026deg;C in 1 L LB medium with appropriate antibiotics. IPTG (final concentration 0.1 mM) was supplemented to the culture when it reached an OD\u003csub\u003e600\u003c/sub\u003e at 0.6\u0026ndash;0.8, and then the induced \u003cem\u003eE. coli\u003c/em\u003e was grown at 11\u0026deg;C for 24 hours. The cells were harvested by centrifugation (5000 rpm, 15 min, 4\u0026deg;C), and resuspended in 20 mL lysis buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH 8.0, 300 mM NaCl, 10 mM imidazole) and lysed by sonication on ice (200 W, 10 s, 10 s, 20 min) with an ultrasonic homogenizer SCIENTZ-IID (SCIENTZ, China). The lysate was centrifugated (12000 g, 1 hour, 4\u0026deg;C), then the supernatant was collected and filtered with a 0.45 \u0026micro;m membrane (Sartorius Stedim Biotech, Germany) to remove residual cellular debris. A nickel affinity chromatography column was prepared with 2 mL Ni-NTA Resin (Trans Gen Biotech, China) loaded into a gravity column (Sangon, China) and used for separation of the filtered supernatant. After washing with wash buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH 8.0, 300 mM NaCl, 20/40/60 mM imidazole), His-tagged proteins were eluted with elution buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH 8.0, 300 mM NaCl, 250 mM imidazole). Purified proteins were concentrated in the storage buffer (50 mM Tris-HCl, pH 7.5, 20% glycerol), and flash frozen. The purity of the protein was confirmed by SDS-PAGE.\u003c/p\u003e \u003cp\u003eThe mutant proteins were purified in a similar procedure with LcsG. Mutated proteins expressions were verified by Western blot using an anti-His antibody (TransGen Biotech Ltd, Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro enzyme assay for LcsG and its mutants\u003c/h2\u003e \u003cp\u003eTo determine the function of LcsG, the enzyme assays (50\u0026micro;L) contained Tris-HCl buffer (50mM, pH 7.5), SAM (2 mM), LeuA or LeuK0 (10 \u0026micro;M), and the purified recombinant LcsG (10 \u0026micro;M). The reactions were incubated at 28\u0026deg;C for 120 min.\u003c/p\u003e \u003cp\u003eFor single-factor experiments, the enzyme assays (50\u0026micro;L) contained Tris-HCl buffer (50mM), SAM (2 mM), PLBJ-1 WT crude extract (200 \u0026micro;g), and the purified recombinant LcsG (10 \u0026micro;M). For the reaction time cause assay, LcsG was tested with Tris-HCl buffer (pH 7.5) at 28\u0026deg;C for 0.01 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min. For the reaction pH cause assay, LcsG was tested at 28\u0026deg;C for 120 min with Tris-HCl buffer in different pH (pH 7.0, pH 7.5, pH 8.0, pH 8.5). For the reaction temperature cause assay, LcsG was tested with Tris-HCl buffer (pH 8.0) for 120 min at 18\u0026deg;C, 21\u0026deg;C, 27\u0026deg;C, 34\u0026deg;C, and 38\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo determine the function of mutated LcsG, 50 \u0026micro;L reaction systems were prepared with 500 nM catalysts, 2 mM SAM, 100 mM Tris-HCl buffer (pH 8.0), and 10 \u0026micro;M substrates. The reactions were incubated at 34\u0026deg;C for 120 min.\u003c/p\u003e \u003cp\u003eThe above enzymatic reactions were quenched by adding 50 \u0026micro;L cold acetonitrile and 10 \u0026micro;L was analyzed by LC-MS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCalibration curves of LeuA0 and LeuK3\u003c/h2\u003e \u003cp\u003eCompounds were quantified by an external standard method and the construction of calibration curves using LeuA0 and LeuK3. In this study, LeuA0 was diluted at concentrations of 25, 50, 100, 250, and 500 nM. LeuK3 was also diluted to five concentrations of 50, 100, 250, 500, to 1000 nM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMichaelis-Menten enzyme kinetics\u003c/h2\u003e \u003cp\u003eKinetic constants of LcsG were determined by the following approach: 1 \u0026micro;M LcsG was assayed against a range of substrate (LeuA or LeuK0) concentrations (2-200\u0026micro;M) with the initial rate of reaction measured by monitoring product formation (LeuA0 or LeuK3) at 34\u0026deg;C for 30 min. The rates were plotted against substrate concentration using the Michaelis-Menten kinetics equation by nonlinear regression analysis with the software GraphPad Prism 8 and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e constants generated from the resulting Michaelis-Menten plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS analysis\u003c/h2\u003e \u003cp\u003eLC-MS analyses were run on an Agilent 1290 Infinity II HPLC with an Agilent Infinity Lab single quadrupole mass selective detector by using an Agilent Zorbax Eclipse Plus C18 reversed-phase column (2.1 \u0026times; 100 mm, 2.7 \u0026micro;m). Water (A) with 0.1% (v/v) formic acid and acetonitrile (B) were used as the solvents at a flow rate of 0.25 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The substances were eluted with 10% (v/v) B for 1 min, then a linear gradient from 10 to 100% (v/v) B in 12 min, washed with 100% solvent B for 5 min, and equilibrated with 5% solvent B for 10 min at a flow rate of 0.25 mL/min. The mass spectrometer was set in electrospray positive ion mode for ionization.\u003c/p\u003e \u003cp\u003eLC-HRESI-MS-MS analyses were run on an Agilent HPLC 1260 Infinity II system equipped with an Agilent G6510A mass spectrometer by using an Agilent Zorbax SB-C18 reversed-phase column (4.6 \u0026times; 150 mm, 5 \u0026micro;m). A linear gradient analytical method (10\u0026ndash;100% MeCN in water with 0.1% formic acid for 20 min at a flow rate of 1.0 mL/min) was used. The Q-TOF was operated in positive mode with a capillary voltage of 1800 V and a drying gas flow rate of 1 \u0026micro;L/min at 300\u0026deg;C. MS scan range was 80\u0026ndash;2000 \u003cem\u003em/z\u003c/em\u003e, and MS-MS scan range was 40\u0026ndash;1400 \u003cem\u003em/z\u003c/em\u003e. Fixed collision energies were 65 V.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial growth inhibition assays\u003c/h2\u003e \u003cp\u003eGrowth inhibition of leucinostatins (LeuK0, LeuK3, LeuA0) with \u003cem\u003eC. neoformans\u003c/em\u003e H99 was assessed on PDA medium with agar diffusion assays. Overnight cultures of \u003cem\u003eC. neoformans\u003c/em\u003e grown in PDB broth at 28\u0026deg;C were diluted with PDB broth to an OD\u003csub\u003e600\u003c/sub\u003e at 0.1. 1 mL aliquots of the resulting mixture were combined with 30 mL aliquots of PDA at 45\u0026deg;C. The test wells (4 mm diameter) were aspirated from the solidified medium using the tip of a sterilized Luer-lock syringe, and 10 \u0026micro;L of compounds were added to the wells. The plate was incubated at 28\u0026deg;C. Zones of inhibition were photographed after 36 hours. Leucinostatins (Solarbio, China) and Amphotericin B were dissolved in DMSO.\u003c/p\u003e \u003cp\u003eGrowth inhibition of leucinostatins (LeuK0, LeuK3, LeuA0) with \u003cem\u003eP. infestans\u003c/em\u003e was assessed on rye agar medium in 9-cm Petri plates. \u003cem\u003eP. infestans\u003c/em\u003e was incubated on the center of plates and cultured at 18\u0026deg;C for 3 d, followed by aspiration test wells (4 mm diam) at the colony edges.\u003c/p\u003e \u003cp\u003eAccording to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations\u003csup\u003e50\u003c/sup\u003e, the MIC (minimal inhibitory concentration) was determined with three replicates using the serial dilution method in 96-well plates with YM (1% maltose extract, 0.2% yeast extract) as the test medium. Amphotericin B was used as the positive control. Test compounds were dissolved in DMSO and serially diluted in a growth medium. Visual endpoint and the optical density readings of microplate wells were measured relative to positive and negative controls. The strains were incubated at 25\u0026deg;C, and the MICs were determined at 48 hours for \u003cem\u003eC. neoformans\u003c/em\u003e H99. Viability was determined with the aid of a plate reader using PrestoBlue resazurin dye (Life Technologies) as the viability indicator. The spectrophotometric MIC value was defined as the lowest concentration of a test compound that resulted in a culture with a density equal to 100% inhibition when compared to the growth of the untreated control.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eStructure prediction of LcsG\u003c/h2\u003e \u003cp\u003eUni-fold was employed to predict LcsG dimer structure. A fasta file of two lcsG sequences was uploaded and a predicted structure was returned. The average pLDDT score was 0.88.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStructure and sequence alignment\u003c/h2\u003e \u003cp\u003eHomology protein structures were searched via HHpred. ChimeraX was used to estimate the structural alignment and plot the figures. Multiple sequences were aligned by mafft, then espript3 was employed to export the sequence alignment results.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eDiffdock\u003c/h2\u003e \u003cp\u003eThe structure of leucinostatin A was modified from the structure of its analogy ZHAWOC6027(PDB: 8a19). The Diffdock webserver was employed to simulate the dock. Predicted LcsG dimer structure and leucinostatin A structure were uploaded and the final docking poses were downloaded. The first rank docking pose was selected as the model.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. Source data are provided with this paper.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the grants from the National Key R\u0026amp;D Program of China (2022YFD1400700), the\u0026nbsp;National Natural Science Foundation of China (32272630)\u0026nbsp;and the\u0026nbsp;Agricultural Science and Technology Innovation Program of CAAS. We also thank Prof. Junfeng Liu and Dr. Xin Zhang (China Agricultural University, China) for their advice on protein purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY, L., B. X, and Z. L. designed the research.\u0026nbsp;Z. L.\u0026nbsp;performed protein purification, fungal fermentation, compounds\u0026nbsp;isolation, structure elucidation, LC-MS analysis, in vivo genetic and in vitro biochemical experiments; Y. J. performed protein purification, LC-MS analysis, in vivo genetic and in vitro biochemical experiments; J. L.\u0026nbsp;and\u0026nbsp;J. Z. performed the genomic analysis; Z. M. and Y. Y. assisted in the test of antimicrobial activity;\u0026nbsp;K. Z. assisted in structure elucidation and chemical synthesis; Z.W. performed LC-MS analysis and LC-HRESI-MS-MS analysis; Y. L., B. X, and Z. L. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting\u0026nbsp;interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSchaner Tooley, C.E., et al.: NRMT is an α-N-methyltransferase that methylates RCC1 and retinoblastoma protein. Nature. \u003cb\u003e466\u003c/b\u003e, 1125\u0026ndash;1128 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStratton, C.F., Poulin, M.B., Du, Q., Schramm, V.L.: Kinetic isotope effects and transition state structure for human phenylethanolamine \u003cem\u003eN\u003c/em\u003e-methyltransferase. ACS Chem. 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Prod. \u003cb\u003e79\u003c/b\u003e, 2357\u0026ndash;2363 (2016)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3280468/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3280468/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eN-\u003c/em\u003emethyltransferase (NMT)-catalyzed methylations are rarely reported at nonribosomal peptides (NRPs) terminuses. Here, we discovered a fungal NMT LcsG for the iterative terminal \u003cem\u003eN\u003c/em\u003e-methyl formation of a family of NRPs, leucinostatins. Gene deletion suggested LcsG is essential to the methylation of leucinostatins. In vitro assay and HRESI-MS-MS analysis proved the methylation sites were the NH\u003csub\u003e2\u003c/sub\u003e, NHCH\u003csub\u003e3\u003c/sub\u003e and N(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e in the C-terminal unit of various leucinostatins. Based on the protein structure predicted by artificial intelligence (AI), molecular docking, and site-directed mutagenesis, we proposed the catalytic mechanism of the LcsG-catalyzed reaction was an N atom coordinated by two negatively charged residues (Asp368, Asp395 for LcsG) towards the subsequent \u003cem\u003eS\u003c/em\u003e\u003csub\u003e\u003cem\u003eN\u003c/em\u003e\u003c/sub\u003e2 methylation. These findings not only provide an approach for enriching the variety of natural bioactivity of NPRs but also deepen the insight into the catalytic mechanism of \u003cem\u003eN\u003c/em\u003e-methylation of NRPs.\u003c/p\u003e","manuscriptTitle":"Characterization of N-methyltransferase for catalyzing the terminus of leucinostatins in Purpureocillium lilacinum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-21 00:07:44","doi":"10.21203/rs.3.rs-3280468/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4f10f6a5-d537-46bc-8790-676852ed42da","owner":[],"postedDate":"September 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":24674534,"name":"Biological sciences/Microbiology/Fungi/Fungal biology"},{"id":24674535,"name":"Biological sciences/Molecular biology/Post-translational modifications/Methylation"}],"tags":[],"updatedAt":"2024-06-23T07:06:12+00:00","versionOfRecord":{"articleIdentity":"rs-3280468","link":"https://doi.org/10.1038/s42003-024-06467-0","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2024-06-22 04:00:00","publishedOnDateReadable":"June 22nd, 2024"},"versionCreatedAt":"2023-09-21 00:07:44","video":"","vorDoi":"10.1038/s42003-024-06467-0","vorDoiUrl":"https://doi.org/10.1038/s42003-024-06467-0","workflowStages":[]},"version":"v1","identity":"rs-3280468","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3280468","identity":"rs-3280468","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}