Subscriber access provided by NEW YORK UNIV
Article
Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains Fumihiro Ishikawa, Sho Konno, Takehiro Suzuki, Naoshi Dohmae, and Hideaki Kakeya ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00097 • Publication Date (Web): 03 Jun 2015 Downloaded from http://pubs.acs.org on June 9, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains
Fumihiro Ishikawa,†,#,* Sho Konno,†,# Takehiro Suzuki,‡ Naoshi Dohmae,‡ and Hideaki Kakeya†,*
†
Department of System Chemotherapy and Molecular Sciences, Division of
Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan ‡
Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science,
2-1 Hirosawa, Wako, Saitama 351-0198, Japan
*
Corresponding authors: Tel: +81-75-753-9267; Fax: +81-75-753-4591; E-mail:
fishika@pharm.kyoto-u.ac.jp
(F.
Ishikawa);
Tel:
+81-75-753-4524;
+81-75-753-4591; E-mail: scseigyo-hisyo@pharm.kyoto-u.ac.jp (H. Kakeya)
ACS Paragon Plus Environment
Fax:
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ABSTRACT Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large diverse families of biosynthetic enzymes that catalyze the synthesis of natural products that display biologically important activities. Genetic investigations have greatly contributed to our understanding of these biosynthetic enzymes; however, proteomic studies are limited. Here we describe the application of active site-directed proteomic probes for adenylation (A) domains to profile the activity of NRPSs directly in native proteomic environments. Derivatization of a 5′-O-N-(aminoacyl)sulfamoyladenosine appended clickable benzophenone functionality enabled activity-based protein profiling of the A-domains in NRPSs in proteomic extracts. These probes were used to identify natural product producing microorganisms, optimize culture conditions, and profile the activity dynamics of NRPSs. Our proteomic approach offers a simple and versatile method to monitor NRPS expression at the protein level and will facilitate the identification of orphan enzymatic pathways involved in secondary metabolite production.
ACS Paragon Plus Environment
Page 2 of 41
Page 3 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
INTRODUCTION Nonribosomal peptides (NRPs) and polyketides (PKs) constitute two large classes of natural products, including clinical antimicrobial, anticancer, and immune suppressive agents, virulence factors, and signaling molecules.1 Because of their broad range of medicinal activities, the enzymes responsible for their production, two large, highly versatile multifunctional enzyme families, known as nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), have been intensely studied in terms of genetic, biochemical, and structural characterization over the past two decades.2,3 These investigations have greatly accelerated the manipulation of natural product biosynthetic pathways to produce new bioactive metabolites through metabolic engineering,4 combinatorial biosynthesis,5 and directed evolution.6 However, our understanding of these biosynthetic enzyme families at the proteomic level remains limited. Little is known regarding the activity dynamics, transcriptional regulation, and posttranslational events of NRPS and PKS proteins in natural product producers. Analogous to the importance of studying the human proteome in the context of the genome, proteomic studies of natural product producer microorganisms provide a level of information that cannot be understood solely by genetic approaches.7 Of the numerous enzyme classes for which a versatile strategy for activity-based protein profiling (ABPP) would be required, NRPS enzymes stand out as particularly important for
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
several reasons. First, these enzyme family members are large and diverse, with numerous members encoded by the bacterial genome.8 These enzymes play a central role in many natural product biosynthetic processes, including the production of not only pharmaceuticals but also small molecule virulence factors.9 Owing to the multifunctionality of the NRPS family and the methodological complications associated with monitoring and predicting NRPS activities in proteomic extracts, there has been great interest in developing methods to assess NRPS activities directly in native proteomic environments. In addition, some of these multifunctional enzymes are particularly resistant to biochemical characterization as intact recombinant proteins. This is partly because these enzymes are large molecular proteins (ranging from 300–800 kDa on average) and because of the general intractability of producer organisms to conventional genetic manipulation and heterologous expression. Profiling these enzyme family members in the proteomes of diverse organisms could prove highly complementary to genetic approaches by allowing us to not only visualize, monitor, and track the activity dynamics of NRPS enzymes, but also facilitate the rapid labeling, enrichment, isolation, and identification of orphan NRPS proteins, as required for proteomic applications. Proteomic Investigation of Secondary Metabolism (PrISM) permits targeted detection of peptide-based natural products produced by NRPs and/or PKs with identification of the gene cluster responsible for synthesis of the natural product.10,11,12,13 Several chemistry-based approaches have exploited profiling thioesterase
ACS Paragon Plus Environment
Page 4 of 41
Page 5 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
(TE),14,15 dehydratase (DH),16 and aryl acid adenylating enzymes17 found in any PKS and/or NRPS enzymes in bacterial proteomes. Conventional strategies to assess the enzymatic activities of the adenylation (A) domains in NRPSs rely on radioactive methods that have been used for over 30 years such as ATP-PPi exchange18,19 and the uptake of radiolabeled amino acids.20,21 These assays pose several laborious, complicated, and time-consuming handling steps involving radioactivity and the labile thioester bond that holds radiolabeled amino acids to the 4′-phosphopantetheine arm of the thiolation (T) domains in NRPS proteins. Other techniques using a continuous hydroxylamine release assay are limited to the analysis of purified proteins.22 Here we describe an alternative strategy that uses active site-directed proteomic probes to covalently label the A-domains in NRPSs in an activity-based manner (Figure 1). Specifically, we expand a set of active site-directed proteomic probes for the A-domains in NRPSs based on derivatization with a 5′-O-N-(aminoacyl)sulfamoyladenosine (aminoacyl-AMS) appended clickable benzophenone functionality, which enables the selective labeling, visualization, enrichment, and identification of NRPS proteins in biological systems. These probes were found to label each A-domain in NRPSs, and proved capable of profiling the activity dynamics of the NRPS/A-domain. In addition, this strategy offers several advantages over conventional methods: (i) enzymes are tested directly in native proteomes, alleviating the need for recombinant expression and
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 41
purification; (ii) active site-directed proteomic probes selectively bind and label the cognate A-domains by ligand-directed protein labeling even if multiple A-domains are housed on large multifunctional megasynthetases; and (iii) in-gel fluorescence scanning has some excellent attributes, including its simplicity, robustness, and nonradioactivity. We chose A-domains in the gramicidin S biosynthetic enzymes, GrsA and GrsB as the initial targets to demonstrate our strategy. This is because gramicidin S was first isolated and characterized antibiotic and thus holds a special place in natural products history. Gramicidin S is
a
cyclic
decapeptide
antibiotic
with
the
primary
structure
cyclo
(-D-Phe1-L-Pro2-L-Val3-L-Orn4-L-Leu5-)2, which is biosynthesized by GrsA and GrsB peptide synthetases (Figure 2). GrsA is derived from gramicidin S biosynthetic enzymes, a single module peptide synthetase containing the domain structure A (L-Phe)-T-E, and responsible for the incorporation of D-Phe during gramicidin S biosynthesis.23 GrsB is a large multifunctional megasynthetase with a calculated molecular weight of 508 kDa, and consists of four NRPS modules, C-A2 (L-Pro)-T-C-A3 (L-Val)-T-C-A4 (L-Orn)-T-C-A5 (L-Leu)-T-TE. Four individual A-domains (A2–A5) are housed on this single protein and selectively incorporate their cognate amino acids, L-Pro, L-Val, L-Orn, and L-Leu, respectively, in the NRPS assembly line.23 The original gramicidin S producing bacterial strain was described as Bacillus brevis var. G.B.24 and deposited as B. brevis ATCC 9999 by the American Type Culture Collection (ATCC). The
ACS Paragon Plus Environment
Page 7 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) conserved this strain as DSM 2895. In addition, DSMZ preserved two other gramicidin S producing strains, B. brevis DSM 5759 and B. brevis DSM 5668. Following gene sequence alignment of 16S rRNA, ATCC 9999 was reclassified as Aneurinibacillus migulanus comb. nov.25 On the basis of DNA-DNA hybridization with DSM 2895, the two other strains, DSM 5759 and DSM 5668, were also assigned to A. migulanus.26 Because of its high stability and simple structure, gramicidin S has been used as not only a reference compound in NMR spectrometry and mass spectrometry but also a model compound for investigating the mechanism of action of membrane-targeting antibiotics.27,28 Gramicidin S had commercially been produced in cultures of A. migulanus. However, the cyclic peptide is no longer available. In addition to strain maintenance, the optimization of culture conditions for nonproducing strains is thus significant in biotechnological applications. RESULTS AND DISCUSSION Design and synthesis of active site-directed proteomic probes for A-domains in NRPSs: An ideal functional proteomics probe for NRPSs would selectively capture this enzyme class in an activity-based manner. With this goal in mind, we targeted A-domains from multiple catalytic components on NRPS enzymes. This is because the A-domain in NRPS is an essential catalytic motif and functions as the gatekeeper of the NRPS assembly line. Briefly, A-domains
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
selectively recognize their cognate amino acids from a much larger monomer pool, including all 20 proteinogenic amino acids as well as a number of nonproteinogenic amino acids, aryl acids, fatty acids, and hydroxy acid building blocks, and convert them to aminoacyl adenylate intermediates at the expense of ATP (Figure 3a).3 The adenylated substrates in turn undergo a nucleophilic attack by the terminal thiol group of the 4′-phosphopantetheine arm of a downstream T domain, forming thioester bound aminoacyl-S-T (Figure 3a). The general design of active site-directed probes for A-domains in NRPSs was based on a tight-binding bisubstrate inhibitor scaffold, aminoacyl-AMS wherein the highly reactive acylphosphate linkage had been substituted by a bioisosteric and chemically stable nonhydrolysable acylsulfamate group.29,30,31,32,33 Accordingly, the amino acid moieties function as warheads, directed to the active sites of A-domains, providing the highly selective binding. We therefore envisaged that aminoacyl-AMS derived probes would exhibit high selectivity for labeling catalytically active adenylating enzymes involved in natural product biosynthesis. As our goal was to generate chemical proteomic probes that could direct the inhibitors towards the active sites of A-domains in NRPSs in a ligand-directed manner, we decided to modify the aminoacyl-AMS scaffold to enable the capture of proximal molecular species to generate covalent enzyme-inhibitor adducts and allow coupling of probe-modified enzymes to reporter tags after the proteome labeling step via Cu(I)-catalyzed azide-alkyne [3+2] click chemistry (CC).34 We elected to modify
ACS Paragon Plus Environment
Page 8 of 41
Page 9 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
aminoacyl-AMS with a clickable benzophenone functionality at the 2′-OH of the adenosine skeleton. Indeed, attachment of the clickable benzophenone linker to L-Phe-AMS 4 and L-Pro-AMS 5 at this position showed no effect on the binding properties of the recombinant
GrsA and TycB1 NRPS enzymes.35 To this end, L-Phe-AMS-BPyne 1,35 L-Val-AMS-BPyne 2, and L-Leu-AMS-BPyne 3 were synthesized by the assembly of the AMS scaffold, the photoreactive benzophenone and the clickable alkyne functionality (Figure 3b and Supplementary Scheme S1). In addition, L-Phe-AMS 4, L-Pro-AMS 5, L-Val-AMS 6, L-Orn-AMS 7, and L-Leu-AMS 8 were prepared to provide analogs of 1–3 for comparative
activity studies.35 Determination of apparent Ki values: Our biochemical studies began with an examination of the specific activities of probe 2 and L-Val-AMS 6 against recombinant AusA1. AusA1 is the excised didomain (A1-T1) of the aureusimine synthetase AusA (A1-T1-C-A2-T2-R) from Staphylococcus aureus.36 The A1 domain of AusA incorporates L-Val during the biosynthesis of aureusimines. Recombinant apo-AusA1 was prepared as a C-terminal His6-tagged fusion protein according to a previously reported procedure.35 Apparent Ki values were determined by a coupled hydroxamate-MesG continuous spectrophotometric assay.22 Reactions contained AusA1 (1.2 µM), Tris pH 8.0 (20 mM), ATP (3 mM), MgCl2 (1 mM), TCEP (1 mM), hydroxylamine pH 7.0 (150 mM), nucleoside phosphorylase
(0.1 U), pyrophosphate (0.04 U),
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
7-methylthioguanosine (MesG; 0.2 mM), and L-Val (60 × Km; 1 mM). Because of the tight-binding characteristics of both probes, the resulting concentration-response plots were fit to the Morrison equation to afford apparent Ki values of 295 ± 16 nM and 212 ± 15 nM for probe 2 and L-Val-AMS 6, respectively (Figure 4). These results validated that modification of the 2′-OH group of the adenosine preserves the high binding affinity of the A-domain of AusA1, suggesting that the aminoacyl-AMS-BPyne scaffold is an optimal design that does not negatively impact on probe-protein interactions. In vitro characterization: Our labeling studies began by examining the ability of probe 2 to selectively label recombinant AusA1. Probe 2 (1 µM) was incubated with AusA1 (1 µM) for 10 min at room temperature and pH 8.0. The sample was then photoactivated with UV light (365 nm) for 60 min at 0 °C, reacted with rhodamine (Rh)-azide (structure shown in Supplementary Figure S1) under standard CC conditions,34 and visualized by SDS-PAGE coupled with in-gel fluorescence imaging. For the inhibition study, AusA1 (1 µM) was preincubated with 6 for 10 min at room temperature. As shown in Figure 5a, labeling of the A-domain of AusA1 was completely abrogated by the addition of the inhibitor, L-Val-AMS 6. This result demonstrated that the labeling reaction mediated by probe 2 was associated with binding in the A-domain active site. We next performed time course studies to investigate the labeling of AusA1. The maximum fluorescence band intensity was observed at 60 min (Figure 5b). The relative labeling
ACS Paragon Plus Environment
Page 10 of 41
Page 11 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
of AusA1 by probe 2 was estimated as 23.5 ± 1.4% at 60 min using a TAMRA-conjugated BSA as a standard of fluorescence intensity (Supplementary Figure S2). To assess the sensitivity of probe 2, AusA1 (1–500 fmol) was treated with probe 2 (1 µM) to determine the limit of detection of AusA1 labeling. We found that probe 2 could detect as low as 25 fmol of AusA1 (Figure 5c). We next asked whether probe 2 could discriminate the cognate A-domain function by ligand-directed protein labeling. TycB1 is the excised tridomain C-A (L-Pro)-T from the multifunctional tyrocidine biosynthetic enzyme, TycB.37 The A-domain of TycB1 incorporates L-Pro during the biosynthesis of tyrocidine. Labeling experiments of GrsA, TycB1, AusA1, and
BSA with probe 2 (1 µM) resulted in selective labeling of the cognate A-domain of AusA1 with no detectable fluorescence signals from TycB1 or BSA (Figure 5d). This finding was identical to our previous results which showed the labeling patterns with L-Phe-AMS-BPyne 1 and L-Pro-AMS-BPyne.
35
In contrast, labeling GrsA by probe 2 showed cross-reactivity with the
A-domain of GrsA with much lower gel scanning intensity than that of labeling the A-domain of AusA1 (Figure 5d). Significantly, the observed signal was completely abrogated by pretreatment of GrsA with L-Phe-AMS 4, suggesting the specific binding of probe 2 to the active site of the GrsA A-domain. This specific cross-reactivity may be attributed to its smaller size compared with L-Phe and the shared hydrophobic property of L-Val. Collectively, the precise active-site chemistry of A-domains allow us to selectively label the A-domain with substrate specificity
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
that corresponds to an attached amino acid moiety. Proteomic characterization: After demonstrating selective labeling of the A-domain of AusA1 by probe 2 in the recombinant enzyme systems, we tested the applicability of probes 2 and 3 by applying them to the analysis of NRPS enzymes from natural product producers. The gramicidin S producing strain, Aneurinibacillus migulanus DSM 5759 was cultured and whole cell lysate was isolated as previously described.35 To evaluate endogenous GrsB labeling, the DSM 5759 proteome (1.5 mg/mL) was individually incubated with probes 2 and 3 (1 µM) for 10 min at room temperature, irradiated for 5 min at 0 °C, and treated with a Rh-azide under CC conditions. In-gel fluorescence scanning showed the labeled fluorescent bands at ~500 kDa and ~100 kDa by probes 2 and 3 (Figures 6a and 6b). Significantly, labeling was inhibited by preincubation with 100 µM of each probe’s cognate competitor (6 and 8; at 100 µM) (Figures 6a and 6b). The high-molecular-weight band in the DSM 5759 proteome has been identified as GrsB.35 To isolate the additional cellular targets of probes 2 and 3, DSM 5759 lysates were individually incubated with probes 2 and 3 in either the absence or presence of individual inhibitors 6 and 8, photo-cross-linked, and subjected to CC with a trifunctional azido-biotin-Rh tag (structure shown in Supplementary Figure S1) followed by streptavidin enrichment. SDS-PAGE of the pulldown eluant revealed bands at ~100 kDa by silver staining (Figures 6c and 6d). Similarly, these bands disappeared after the addition of 6 and 8 (Figures 6c and 6d). Samples of the
ACS Paragon Plus Environment
Page 12 of 41
Page 13 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
excised bands were submitted for LC-MS/MS protein identification and found to contain a protein highly homologus to valine-tRNA ligase (valyl-tRNA synthetase, ValRS) from Aneurinibacillus aneurinilyticus with 48% peptide coverage and the leucine-tRNA ligase (leucyl-tRNA synthetase, LeuRS) from A. aneurinilyticus with 27% peptide coverage, respectively (Supplementary Figures S6, S7, S11, and S12). Aminoacyl-AMS molecules have been used in structural studies on a number of aminoacyl-tRNA synthetases (aaRSs).38,39 Taken together, these results confirmed that the aminoacyl-AMS-BPyne scaffold is sensitive to a shared catalytic mechanism, namely, the formation of an enzyme-bound aminoacyl-adenylate monophosphate by the A-domains in NRPSs and aaRSs. Using probe molecules 2 and 3, we next attempted to demonstrate selective labeling of the A-domains housed on GrsB by a combination of these probes and inhibitors 4–8. As shown in Figure 6e, the labeling of GrsB by 2 was abrogated by preincubation with either 6 or 8. In contrast, the labeling of GrsB by probe 3 disappeared only after the addition of 8. These labeling patterns confirmed that probes 2 and 3 selectively targeted individual A-domains housed on GrsB, A3 (L-Val) and A5 (L-Leu), respectively. Concurrently, the competitive profiling revealed that 8 could bind to not only A4 (L-Leu) domain, but also to the A3 (L-Val) domain because of the lack of labeling of GrsB by probe 2. The L-Val activating A-domain of AusA is known to recognize and activate L-Leu, L-Ile, L-cycloleucine, L-Ala, L-Cys, and tert-butylleucine as
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
miscognate substrates with lower catalytic efficiency than that of the cognate substrate, L-Val.36 The specificity conferring code of the L-Val activating A-domain in GrsB is identical to that of AusA, except for Ile299, which is replaced with Leu in the A-domain of AusA (Supplementary Table S1). The A-domain of GrsB could therefore share similar substrate preferences to the A-domain of AusA. To investigate endogenous GrsA labeling, probe 1 (1 µM) was added to DSM 5759 proteomes (2.0 mg/mL) in the absence or presence of 4 (100 µM). Following a 10-min incubation, proteomes were photoactivated with UV light for 30 min, treated with an azide-conjugated Rh tag under CC conditions, and resolved by SDS-PAGE. In-gel fluorescence scanning identified an intensely labeled protein with a molecular mass of ~120 kDa corresponding to the size of endogenous GrsA (Figure 7a). In addition, the observed single band was competitively inhibited in the presence of 4. To confirm the cellular targets of probe 1, we next pursued the molecular characterization of the labeled protein in the DSM 5759 proteome. The DSM 5759 proteome was incubated with probe 1 in the absence or presence of 4, photo-cross-linked for 30 min, and subjected to CC with a trifunctional azido-biotin-Rh tag followed by streptavidin enrichment. SDS-PAGE of the pulldown eluant was visualized by silver staining, and the 120 kDa band was excised, subjected to in-gel trypsin digestion, analyzed by LC-MS/MS, and indeed found to contain endogenous GrsA with 23% peptide
ACS Paragon Plus Environment
Page 14 of 41
Page 15 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
coverage (Supplementary Figures S8 and S13). NRPS activity profiles of the proteomes of natural product producing bacteria: A comprehensive examination of NRPS activities would yield proteomic information of sufficient quantity and quality to portray higher-order natural product producing bacterial properties. We next asked whether probes 1 and 3 could be used to profile NRPS enzyme activities in bacterial proteomes. Four strains of A. migulanus which have been deposited as producers of gramicidin S were evaluated with regard to their quantitative yield of peptide biosynthesis. Berditsch and co-workers demonstrated that only ATCC 9999 and DSM 5759 were able to biosynthesize the cyclic peptide, whereas DSM 2895 and DSM 5668 provided zero yield using a sporulation-promoting NBYS medium and an AN-rich YP medium (Supplementary Table S2).40 Our first challenge was to optimize fermentation conditions for the nonproducing strains, DSM 5668 and DSM 2895. We thus decided to cultivate four bacteria using a complex YPG medium. This medium is described as one of the most effective complex media for gramicidin S biosynthesis and is composed of yeast extract, peptone, and glucose.41 The four strains of A. migulanus were cultured in YPG medium for 24 h at 37°C, reaching OD660 values of 30.0 (ATCC 9999), 15.0 (DSM 5759), 17.4 (DSM 5668), and 11.2 (DSM 2895). To investigate GrsA activity, probe 1 (1 µM) was added to individual bacterial proteomes (2.0 mg/mL) in the absence or presence of 4 (100 µM). In-gel fluorescence imaging identified a labeled protein
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
with a molecular mass of ~120 kDa in the producing strains, ATCC 9999 and DSM 5759. In contrast, no specific labeled proteins were observed in the DSM 2895 proteome. Significantly, a very weak signal was observed in the DSM 5668 proteome at approximately 120 kDa corresponding to the size of endogenous GrsA, suggesting that the labeled protein in the DSM 5668 proteome could represent GrsA. To confirm the expression of active GrsB in the proteomes, probe 3 (1 µM) was incubated with each proteome (2.0 mg/mL) in the absence or presence of 8. Labeling of the proteomes with probe 3, followed by photolysis for 5 min, CC reaction with a Rh-azide tag, SDS-PAGE, and in-gel fluorescence scanning, revealed clear fluorescent signals at approximately 500 kDa in the proteomic samples of ATCC 9999, DSM 5759, and DSM 5668, but not DSM 2895 (Figure 7b). Preincubation of the ATCC 9999, DSM 5759, and DSM 5668 proteomes with 8 resulted in strong inhibitor-sensitive labeling of ~500 kD proteins. Inspection of the gene cluster encoding gramicidin S synthetases led us to identify these proteins as GrsB on the basis of their molecular weight and the presence of the A-domain for L-Leu. Band excision and MS/MS analysis identified endogenous GrsB protein with 36% (ATCC 9999) and 48% (DSM 5668) peptide sequence coverage (Supplementary Figures S9, S10, S14, and S15). We then evaluated their quantitative yield of peptide biosynthesis using high performance liquid chromatography (HPLC). Because of the intracellular localization of gramicidin S,42 the cultures were grown to appropriate OD660 values then immediately
ACS Paragon Plus Environment
Page 16 of 41
Page 17 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
centrifuged, and gramicidin S was extracted from the pellet, as previously reported.40 Quantitative analysis revealed that strains ATCC 9999, DSM 5759, and DSM 5668 could produce the cyclic peptide, with a calculated yield of 880 mg/L, 1580 mg/L, and 680 mg/L, respectively, during 24 h fermentation (Figure 7c). Indeed, ATCC 9999 and DSM 5759 bacteria expressed higher levels of active NRPSs, GrsA, and GrsB than those of DSM 5668. These results correlated well with the expression levels of active GrsA and GrsB proteins at 24 h in producer organisms, allowing us to confirm that these are natural product producing microorganisms and to verify the optimization of bacterial culture conditions. Using the AN-rich YPG medium, ATCC 9999 and DSM 5759 displayed 6- and 15-fold and 2- and 2-fold higher absolute yields of gramicidin S than in a sporulation-promoting NBYS and an AN-rich YP medium, respectively (Supplementary Table S2). It is noteworthy that while DSM 5668 provided no yield in both NBYS and YP media, replacing NBYS and YP with YPG allowed us to deregulate and switch on a silent gramicidin S biosynthetic pathway in DSM 5668. As a result of this induction, we were able to visualize and identify two catalytically active NRPS proteins, GrsA and GrsB in DSM 5668 proteomes. In contrast, while ATCC 9999 and DSM 2895 were thought because of their origin to be absolute equivalents, DSM 2895 did not show any significant gramicidin S production even in YPG medium by HPLC. This finding corresponded to the observed lack of labeling of GrsA and GrsB enzymes in DSM 2895
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
proteomes. These findings highlight that chemical proteomic methods can detect differences in NRPS enzyme activities with a level of accuracy and sensitivity compatible with profiling low abundance proteins in complex proteomes. Despite advances in genomic and proteomic methods, these approaches primarily measure changes in transcript and protein abundance and offer only an indirect readout of the dynamics of protein activities. Therefore, little is known about the activities, protein–protein interactions, transcriptional regulation, and posttranslational events of biosynthetic enzymes in natural product producer organisms, that ultimately would provide insight into the natural product producing behavior of bacteria. To address these limitations, we describe the application of a chemical proteomics strategy to quantitatively monitor NRPS enzyme activities across natural product production processes. The highly producing strain DSM 5759 was cultured in YPG medium at 37°C for the indicated time and 660 nm absorbance, then whole cellular lysates were prepared to a final concentration of 2.0 mg/mL. To understand the activity patterns of GrsA, individual proteomes were incubated with 1 µM of probe 1 in either the absence or presence of 100 µM of 4. Proteomes were irradiated for 30 min, treated with an azide-conjugated Rh tag under CC conditions and resolved by SDS-PAGE. In-gel fluorescence scanning displayed time-dependent labeling of GrsA upon increasing the gel scan intensity (Figure 7d). Monitoring the enzymatic activity of GrsB by 1 µM of probe 3, followed by UV photolysis for 5 min, CC
ACS Paragon Plus Environment
Page 18 of 41
Page 19 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
reaction with a Rh-azide tag, SDS-PAGE, and in-gel fluorescence imaging, revealed maximum labeling of GrsB at 16 h followed by decreased labeling with increasing time (Figure 7e). While the genes grsA and grsB are organized in an operon and transcribed unidirectionally as one polycistronic transcriptional unit,23 these studies have highlighted the diverse patterns of enzyme activities of GrsA and GrsB in DSM 5759 proteomes. The enzymatic activity of GrsA was detected abruptly at 16 h after inoculation. In contrast, GrsB labeling by probe 3 could be observed as a distinguishable fluorescence signal from 12 to 24 h. However, the levels of activity of the megasynthetase of GrsB decreased with increasing time from 20 to 24 h. This may be because of either the unanticipated post-lysis proteolytic events or the lability of endogenous GrsB protein. To evaluate the correlation between enzyme activity and absolute yield of gramicidin S, 1 mL of culture was removed from the flask every 4 h after the culture reached an OD660 value of 2.0 (12 h) in YPG medium. HPLC quantification revealed a time-dependent yield of gramicidin S of an estimated 0.15 mg/mL at 12 h (OD660 = 2.0), 0.24 mg/mL at 16 h (OD660 = 6.0), 1.19 mg/mL at 20 h (OD660 = 15.0), and 1.58 mg/mL at 24 h (OD660 = 20) (Figure 7f). The production of gramicidin S was initiated at ~12 h of cultivation, at an OD660 value of ~2.0, indicating that production fully correlated with increasing cell density. While the gramicidin S yield could be quantified at the early exponential phase (12 h), the fluorescent signal for GrsA in the proteome sample was not observed, indicating an
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
undetectable level of active GrsA at this time point. Gramicidin S was rapidly synthesized during the exponential phase after 16 to 20 h, reaching an OD660 of 6.0 to 15.0, this corresponded to the appearance of active GrsA and maximum enzymatic activity of GrsB at 16 h. These results suggested that chemical proteomic strategies would advance our knowledge of natural product biosynthesis by providing insight into the dynamic and regulation processes of NRPS activities in the context of wild-type producers and will guide future efforts to identify and characterize important biosynthetic pathways at the proteomic level. In addition, the ability to visualize and quantify NRPS activities directly in proteomic environments, where the dynamic processes that regulate NRPS function are reflected, may be applied in cases where the cloning or expression of NRPSs has been unsuccessful. It may be possible to directly characterize key mechanistic steps in NRP and PK-NRP biosynthetic assembly lines. CONCLUSION Here we have demonstrated that A-domain directed ABPP probes will serve as valuable chemical proteomic tools for the functional characterization of NRPSs, a large and diverse enzyme family. In addition to their application in the proteomic profiling of NRPS activities, these studies highlight that proteomic strategies (i.e., ABPP using the labeling of the A-domains in NRPSs) are capable of generating active molecular profiles that accurately detect and characterize natural product producing behavior in bacteria. Furthermore, ABPP is a simple,
ACS Paragon Plus Environment
Page 20 of 41
Page 21 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
rapid, and sensitive method for the comparative characterization of proteomic samples, allowing us to analyze a number of bacteria under a variety of experimental conditions in parallel and profile the dynamics of the activities of NRPS enzymes. In addition, chemical proteomic probes that target ubiquitous NRPSs would prove valuable research tools for the functional characterization of NRPS enzymes and the identification of orphan NRPSs and biosynthetic pathways involved in the construction of peptide-based natural products in a wide range of bacterial systems. ACKNOWLEDGMENTS This work was partly supported by a Grant-in Aid for Young Scientists (B) 26750370 (F.I.), and research grants from the Japan Society of the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) (H.K.). We thank Prof. M. Marahiel (Philipps-Universität Marburg, Germany) for providing the GrsA and TycB1 expression constructs. SUPPORTING INFORMATION Supplemental
figures;
procedures for
the
syntheses of
L-Val-AMS-BPyne
2
and
1
L-Leu-AMS-BPyne 3; complete gel images; full experimental details; and copies of H and 13
C-NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
NOTES
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The authors declare no competing financial interest. #
These authors contributed equally to this work.
REFERENCES (1) Walsh, C. T. (2004) Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810. (2) Fischbach, M. A. and Walsh, C. T. (2006) Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem. Rev. 106, 3468– 3496. (3) Sieber, S. A. and Marahiel, M. A. (2005) Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics. Chem. Rev. 105, 715–738. (4) Tang, L., Shah, S., Chung, L., Camey, J., Katz, L., Khosla, C., and Julien, B. (2000) Cloning and heterologous expression of the epothilone gene cluster. Science 287, 640–642. (5) Nguyen, K. T., Ritz, D., Gu, J.-Q., Alexander, D., Chu, M., Miao, V., Brian, P., and Baltz, R. H. (2006) Combinatorial biosynthesis of novel antibiotics related to daptomycin. Proc. Natl. Acad. Sci. USA 103, 17462–17467. (6) Evans, B. S., Chen, Y., Metcalf, W. W., Zhao, H., and Kelleher, N. L. (2011) Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo. Chem. Biol. 18, 601–607.
ACS Paragon Plus Environment
Page 22 of 41
Page 23 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
(7) Reyes, C. P., La Clair, J. J., and Burkart, M. D. (2010) Metabolic probes for imaging endosymbiotic bacteria within toxic dinoflagellates. Chem. Commun. 46, 8151–8153. (8) Stein, T. (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 56, 845–857. (9) Cisar, J. S. and Tan, D. S. (2008) Small molecule inhibition of microbial natural product biosynthesis – An emerging antibiotic strategy. Chem. Soc. Rev. 37, 1320–1329. (10) Bumpus, S. B., Evans, B. S., Thomas, P. M., Ntai, I., and Kelleher, N. L. (2009) A proteomics approach to discovering natural products and their biosynthetic pathways. Nat. Biotechnol. 27, 951–956. (11) Evans, B. S., Ntai, I., Chen, Y., Robinson, S. J., and Kelleher, N. L. (2011) Proteomics-based discovery of koranimine, a cyclic imine natural product. J. Am. Chem. Soc. 133, 7361–7319. (12) Chen, Y., Ntai, I., Ju, K. S., Unger, M., Zamdborg, L., Robinson, S. J., Doroghazi, J. R., Labeda, D. P., Metcalf, W. W., and Kelleher, N. L. (2012) A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria. J. Proteome Res. 11, 85–94. (13) Chen, Y., Unger, M., Ntai, I., McClure, R. A., Albright, J. C., Thomson, R. J., and Kelleher, N. L. (2013) Gobichelin A and B: mixed-ligand siderophores discovered using proteomics. Med. Chem. Commun. 4, 233–238
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(14) Meier J. L., Niessen, S., Hoover, H. S., Foley, T. L., Cravatt, B. F., Burkart, M. D. (2009) An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis. ACS Chem. Biol. 4, 948–957. (15) Meier, J. L. and Burkart, M. D. (2010) Proteomic analysis of polyketide and nonribosomal peptide biosynthesis. Curr. Opin. Chem. Biol. 15, 1–9. (16) Ishikawa, F., Haushalter, R. W., Burkart, M. D. (2012) Dehydratase-specific probes for fatty acid and polyketide synthases. J. Am. Chem. Soc. 134, 769–772. (17) Duckworth, B. P., Wilson, D. J., Nelson, K. M., Boshoff, H. I., Barry, C. E. III, Aldrich, C. C. (2012) Development of a selective activity-based probe for adenylating enzymes: profiling MbtA involved in siderophore biosynthesis from Mycobacterium tuberculosis. ACS Chem. Biol. 7, 1653–1658. (18) Matteo, C. C., Glade, M., Tanaka, A., Piret, J., and Demain, A. L. (1975) Microbiological studies on the formation of gramicidin S synthetases. Biotechnol. Bioeng. 17, 129–142. (19) Otten, L. G., Schaffer, M. L., Villiers, B. R. M., Stachelhaus, T., and Hollfelder, F. (2007) An optimized ATP/PP(i)-exchangeassay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis. (2007) Biotechnol. J. 2, 232–240. (20) Lee, S. G. and Lipmann, F. (1975) Tyrocidine synthetase system. Methods Enzymol. 43, 585–602.
ACS Paragon Plus Environment
Page 24 of 41
Page 25 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
(21) Chen, H., Hubbard, B. K., O’Connor, S. E., and Walsh, C. T. (2002) Formation of beta-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chem. Biol. 9, 103–112. (22) Wilson, D. J. and Aldrich, C. C. (2010) A continuous kinetic assay for adenylation enzyme activity and inhibition. Anal. Biochem. 404, 56–63. (23) Kratzschmar, J., Krause, M., and Marahiel, M. A. (1989) Gramicidine S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J. Bacteriol. 171, 5422–5429. (24) Gause, G. F. and Brazhnikova, M. G. (1994) Gramicidin S. Origin and mode of action. Lancet 1944, 247, 715–716. (25) Shida, O., Takagi, K., Kadowaki, K., and Komagata, K. (1996) Proposal for two new genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. Int. J. Syst. Bacteriol. 1996, 46, 939–946. (26) Goto, K., Fujita, R., Kato, M., Asahara, M., and Yokota, A. (2004) Reclassification of Brevibacillus brevis NCIMB 13288 and DSM 6472 (= NRRL NRS-887) as Aneurinibacillus danicus sp. nov. and Brevibacillus limnophilus sp. nov. Int. J. Syst. Evol. Microbiol. 54, 419– 427. (27) Hiraoka, K., Mori, K., and Asakawa, D. (2006) Fundamental aspects of electrospray droplet impact/SIMS. J. Mass. Spectrom. 41, 894–902.
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(28) Ulrich, A. S. (2005) Solid state 19F NMR methods for studying biomembranes. Prog. Nucl. Magn. Reson. Spectrosc. 46, 1–21. (29) Isono, K., Uramoto, M., Kusakabe, H., Miyata, N., Koyama, T., Ubukata, M., Sethi, S. K., McCloskey, J. A. (1984) Ascamycin and dealanylascamycin, nucleoside antibiotics from Streptomyces sp. J. Antibiot. 37, 670–672. (30) Ueda, H., Shoku, Y., Hayashi, N., Mitsunaga, J., In, Y., Doi, M., Inoue, M., Ishida, T. (1991) X-ray crystallographic conformational study of 5’-O-[N-(L-alanyl)-sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase. Biochim. Biophys. Acta. 1080, 126–134. (31) Finking, R., Neumüller, A., Solsbacher, J., Konz, D., Kretzschmar, G., Schweitzer, M., Krumm, T., and Marahiel, M. A. (2003) Aminoacyl adenylate substrate analogues for the inhibition of adenylation domains of nonribosomal peptide synthetases, ChemBioChem 4, 903– 906. (32) Ferreras, J. A., Ryu, J.-S., Di Lello, F., Tan, D. S., and Quadri, L. E. N. (2005) Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis. Nat. Chem. Biol. 1, 29–32. (33) Qiao, C., Gupte, A., Boshoff, H. I., Wilson, D. J., Bennett, E. M., Somu, R. V., Barry, III, C. E., and Aldrich, C. C. (2007) 5’-O-[(N-acyl)sulfamoyl]adenosines as antitubercular agents that inhibit MbtA: an adenylation enzyme required for siderophore biosynthesis of the mycobactins,
ACS Paragon Plus Environment
Page 26 of 41
Page 27 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
J. Med. Chem. 50, 6080–6094. (34) Speers, A. E., Adam, G. C., and Cravatt, B. F. (2003) Activity-based protein profiling in vivo using a copper (i)-catalyzed azide-zlkyne [3+2] cycloaddition. J. Am. Chem. Soc. 125, 4686–4687. (35) Konno, S., Ishikawa, F., Suzuki, T., Dohmae, N., Burkart, M. D., and Kakeya, H. (2015) Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases. Chem.Commun. 51, 2262–2265. (36) Wilson, D. J., Shi, C., Teitelbaum, A. M., Gulick, A. M., and Aldrich, C. C. (2013) Characterization of AusA: A dimodular nonribosomal peptide synthetase responsible for production of aureusimine pyrazinones. Biochemistry 52, 926–937. (37) Mootz, H. D. and Marahiel, M. A. (1997) The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J. Bacteriol. 179, 6843–6850. (38) Belrhali, H., Yaremchuk, A. D., Tukalo, M. A., Larsen, K., Berthet-Colominas, C., Leberman, R., Beijer, B., Sproat B, Als-Nielsen, J., Grübel, G., Legrand, J-F., Lehmann, M., and Cusack, S. (1994) Crystal structures at 2.5Å resolution of seryl-tRNA synthetase complexed with two analogues of seryl-adenylate. Science 263, 1432–1436. (39) Cusack, S., Yaremchuk, A., and Tukalo, M. (2000) The 2Å crystal structure of leucyl-tRNA
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
synthetase and its complex with a leucyl-adenylate analogue. EMBO J. 19, 2351–2361. (40) Berditsch, M., Afonin, S., and Ulrich, A. S. (2007) The ability of Aneurinibacillus migulanus (Bacillus brevis) to produce the antibiotic gramicidin S is correlated with phenotype variation. Appl. Environ. Microbiol. 73, 6620–6628. (41) Augenstein, D. C., Thrasher, K. D., Sinskey, A. J., and Wang, D. I. C. (1974) Optimization in the recovery of a labile intracellular enzyme. Biotechnol. Bioeng. 16, 1433–1447. (42) Matteo, C. C., Glade, M., Tanaka, A., Piret, J., Demain, A. L. (1975) Microbiological studies on the formation of gramicidin S synthetases. Biotechnol. Bioeng. 17, 129–142.
ACS Paragon Plus Environment
Page 28 of 41
Page 29 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
FIGURE LEGENDS
Figure 1. Functional proteomic analysis of nonribosomal peptide synthetase (NRPS) activities using active site-directed proteomic probes for adenylation (A) domains. Modules are comprised of thiolation (T), adenylation (A: A1–A4), epimerization (E), condensation (C), and thioesterase (TE) domains. The rectangles represent nonspecific proteins. In gel-based analysis, recombinant NRPS enzymes or proteomes treated with probe molecules are incubated with a rhodamine (Rh)-azide reporter tag under click chemistry (CC) conditions34 and separated by SDS-PAGE, and labeled proteins are visualized by in-gel fluorescence imaging. In addition, proteomes are treated with probe molecules and reacted with a trifunctional azido-biotin-Rh tag under CC conditions, followed by streptavidin enrichment, digestion with trypsin, and analysis by LC-MS/MS.
Figure 2. Nonribosomal peptide synthesis of the antibiotic gramicidin S. Modules are comprised of thiolation (T), adenylation (A) [A1: L-Phe; A2: L-Pro; A3: L-Val; A4: L-Orn; A5: L-Leu specific A-domains], epimerization (E), condensation (C), and thioesterase (TE) domains.
Figure 3. (a) Enzyme mechanism catalyzed by the adenylation (A) domains in nonribosomal
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 30 of 41
peptide synthetases (NRPSs). (b) General structure of the clickable alkyne-tagged 5′-O-N-(aminoacyl)sulfamoyladenosine
(aminoacyl-AMS)
probes
1–3
and
5′-O-N-(aminoacyl)sulfamoyladenosine (aminoacyl-AMS) inhibitors 4–8 for the proteome profiling of NRPS activities.
Figure 4. Concentration-response plot of the fractional initial velocity of the adenylation reaction catalyzed by AusA1 as a function of L-Val-AMS-BPyne 2 (a) and L-Val-AMS 6 (b). Reactions contained apo-AusA1 (1.2 µM), L-Val (1 mM), and standard assay buffer [20 mM Tris (pH 8.0), 3 mM ATP, 1 mM MgCl2, 1 mM TCEP, 150 mM hydroxylamine (pH 7.0), 0.1 U purine nucleoside phosphorylase, 0.04 U inorganic pyrophosphatase, and 0.2 mM MesG]. The curve represents the best nonlinear fit of the data to the Morrison equation. The data points represent the mean with the standard error of duplicate experiments.
Figure 5. Labeling of recombinant apo-AusA1 with probe 2. (a) Labeling of AusA1 and competitive inhibition study with excess inhibitor 6. AusA1 (1 µM) was pre-incubated in either the absence or presence of 100 µM of inhibitor 6 and treated with 1 µM of probe 2. (b) UV photolysis time analysis of the labeling of AusA1 with probe 2. SDS-PAGE analysis depicting the labeling of AusA1 (1 µM) with probe 2 (1 µM). (c) Sensitivity of detection of AusA1
ACS Paragon Plus Environment
Page 31 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
labeling. AusA1 (1–500 fmol) was treated with probe 2 (1 µM). (d) Labeling property of probe 2. GrsA (1 µM), TycB1 (1 µM), AusA1 (1 µM), and BSA (1 µM) were incubated with probe 2 (1 µM) in either the absence or presence of inhibitors 4, 5, and 6 (100 µM). For each panel, Φ depicts the fluorescence observed with λex = 532 nm and λem = 580 nm and Σ displays the total protein content as determined by staining with Coomassie blue (a, b, and d) or silver (c). Full gels (see Supplementary Figure S3) are provided in the Supporting Information.
Figure 6. Proteomic characterization of L-Val-AMS-BPyne 2 and L-Leu-AMS-BPyne 3. (a, b) Labeling of endogenous GrsB in the A. migulanus DSM 5759 cellular lysate by probes 2 and 3. The A. migulanus DSM 5759 lysate (1.5 mg/mL) was individually treated with 6 and 8 (100 µM) before the addition of probes 2 and 3 (1 µM). (c, d) Pull-down assay of probe-binding proteins in the bacterial proteome. The A. migulanus DSM 5759 proteomes (1.0–1.4 mg/mL) were treated with probes 2 or 3. Control samples were preincubated with 100 µM of each competitor (6 or 8). Samples were photo-cross-linked and subjected to CC with biotin-azide followed by streptavidin enrichment. Binding proteins were eluted by boiling in SDS-loading buffer, subjected to SDS-PAGE, and detected with silver stain followed by tryptic digestion and LC-MS/MS analysis. (e) Individual labeling of NRPS A-domains using a combination of probes 1 and 2 and inhibitors 4–8. The A. migulanus DSM 5759 lysate (1.5 mg/mL) was treated with
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
inhibitors 4–8 individually (100 µM) before the addition of 1 µM of probes 2 and 3. For each panel, Φ depicts the fluorescence observed with λex= 532 nm and λem = 580 nm and Σ displays the total protein content as determined by staining with Coomassie blue (a and b) or silver (c and d). Full gels (see Supplementary Figure S4) are provided in the Supporting Information.
Figure 7. NRPS activity profiles of the proteomes of A. migulanus strains. (a) In-gel fluorescence analysis of the GrsA activity profiles obtained from reactions between the proteomes of A. migulanus strains and probe 1. Proteomes (2.0 mg/mL) were treated with probe 1 (1 µM) in either the absence or presence of 4 (100 µM). An arrowhead points to the endogenous GrsA. (b) In-gel fluorescence analysis of the GrsB activity profiles of the A. migulanus proteomes labeled by probe 3. Proteomes (2.0 mg/mL) were incubated with probe 3. Control samples were preincubated with 8 (100 µM). An arrowhead points to the endogenous GrsB. (c) Corresponding absolute (mg/mL) production of gramicidin S. The cultures were cultivated in YPG medium. Gramicidin S yields were determined at 24 h after inoculation. (d) Visualizing the expression of active GrsA in DSM 5759 proteomes by probe 1 at the times indicated. Proteomes (2.0 mg/mL) were incubated with probe 1 (1 µM). Controls were preincubated with 4 (100 µM). (e) Monitoring the GrsB activity in DSM 5759 proteomes by probe 3 at the times indicated. Proteomes (2.0 mg/mL) were reacted with probe 3 (1 µM) in
ACS Paragon Plus Environment
Page 32 of 41
Page 33 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
either the absence or presence of 8 (100 µM). (f) Absolute yield (mg/mL) of gramicidin S, monitored as a function of time for A. migulanus DSM 5759 in YPG medium. For each panel, Φ depicts the fluorescence observed with λex= 532 nm and λem = 580 nm and Σ displays the total protein content as determined by staining with Coomassie blue. Full gels (see Supplementary Figure S5) are provided in the Supporting Information.
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figures
Figure 1.
ACS Paragon Plus Environment
Page 34 of 41
Page 35 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Figure 2.
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 3.
ACS Paragon Plus Environment
Page 36 of 41
Page 37 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Figure 4.
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 5.
ACS Paragon Plus Environment
Page 38 of 41
Page 39 of 41
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
Figure 6.
ACS Paragon Plus Environment
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 7.
ACS Paragon Plus Environment
Page 40 of 41
Page 41 of 41
ACS Chemical Biology
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Paragon Plus Environment