Functional Characterization of the Unique Terminal Thioesterase

Article pubs.acs.org/biochemistry

Functional Characterization of the Unique Terminal Thioesterase Domain from Polymyxin Synthetase Charles A. Galea,† Kade D. Roberts,† Yan Zhu,§ Philip E. Thompson,‡ Jian Li,*,†,§ and Tony Velkov*,† †

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences and ‡Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia § Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Parkville, Victoria 3800, Australia S Supporting Information *

ABSTRACT: Polymyxins remain one of the few antibiotics available for treating antibiotic resistant bacteria. Here we describe polymyxin B thioesterase which performs the final step in polymyxin B biosynthesis. Isolated thioesterase catalyzed cyclization of an Nacetylcystamine polymyxin B analogue to form polymyxin B. The thioesterase contained a catalytic cysteine unlike most thioesterases which possess a serine. Supporting this, incubation of polymyxin B thioesterase with reducing agents abolished enzymatic activity, while mutation of the catalytic cysteine to serine significantly decreased activity. NMR spectroscopy demonstrated that uncyclized polymyxin B was disordered in solution, unlike other thioesterase substrates which adopt a transient structure similar to their product. Modeling showed the thioesterase substrate-binding cleft was highly negatively charged, suggesting a mechanism for the cyclization of the substrate. These studies provide new insights into the role of polymyxin thioesterase in polymyxin biosynthesis and highlight its potential use for the chemoenzymatic synthesis of polymyxin lipopeptides.

T

where each module adds a single building block to the growing polypeptide chain as it is passed from module to module (Figure 1A). The core domains that comprise a module include the adenylation, condensation, and peptidyl carrier domains.9,13,16 The adenylation domain catalyzes a two-step reaction that activates the amino acyl substrate and transfers the amino acid to the thiol of the pantetheine cofactor of the peptidyl carrier protein domain. Located at the N-terminus of most modules, the condensation domain catalyzes amide bond formation. The termination module contains a C-terminal type I thioesterase (TE) domain (25−35 kDa) which catalyzes the thioester cleavage and release of the fully assembled peptide. TE domains commonly contain a canonical catalytic triad consisting of a Ser, His, and an acidic residue.16−18 Sequential acylation and deacylation of the active-site serine result in cleavage of the thioester bond and release of the product. During the release stage, the full-length polypeptide is transferred from the upstream peptidyl carrier domain to the TE catalytic serine residue in the acylation step.9,19,20 Deacylation of the resulting acyl-O-TE intermediate then occurs via intramolecular cyclization or hydrolysis.9,19,20 Most NRPS TE domains catalyze cyclization rather than the hydrolytic cleavage of the polypeptide product.18,19 This leads to the release of bioactive cyclic products; notable examples include daptomycin, tyrocidine, or lipid-containing cyclic

he increasing worldwide prevalence of antibiotic-resistant bacteria, together with the lack of new antibiotics in the development pipeline, threatens the effective prevention and treatment of a wide range of bacterial infections. Initially discovered more than 60 years ago,1 polymyxins have reemerged in recent times as a last-resort class of antibiotics for the treatment of multidrug resistant bacterial infections.2 The development of polymyxin resistance and recent emergence of MCR plasmid-mediated polymyxin resistance have stimulated the discovery of novel polymyxins with better antibacterial activity and reduced nephrotoxicity.3 Polymyxins are lipopeptides comprised of a cyclic heptapeptide loop with an exocyclic tripeptide sequence acylated by an N-terminal fatty acid chain.4 The intramolecular cyclic heptapeptide loop is linked between the side chain of a diaminobutyric acid (Dab) residue at position 4 and the backbone carboxyl group of the Cterminal L-threonine residue at position 10. Polymyxins contain several Dab residues that are positively charged at physiological pH making them polycationic.4 Over 30 structurally distinct polymyxins have been isolated, and they all share similar structures varying only at the N-terminus and positions 3, 6, 7, and 10 within the polypeptide chain. Microbial polyketide synthases (PKSs), nonribosomal peptide synthases (NRPSs), and mixed PKS/NRPS complexes synthesize a vast array of natural products, including important antibiotics such as polymyxins, daptomycin, and vancomycin.5−11 NRPSs are large, multifunctional proteins comprised of distinct modules containing catalytic domains involved in the initiation, chain elongation, modification, and termination of peptide synthesis.9,11−15 Essentially, they act as an assembly line © XXXX American Chemical Society

Received: November 9, 2016 Revised: January 6, 2017 Published: January 10, 2017 A

DOI: 10.1021/acs.biochem.6b01139 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Schematic of the PMB nonribosomal protein synthase from P. polymyxa (ATCC 10401). (A) The synthase subunits PmxA, pmxB, and PmxE are represented by boxes and comprise of one or more modules which activate and incorporate one amino acid residue. Each module is comprised of several domains: C, condensation; A, adenylation; E, epimerization; PCP, peptidyl carrier; T, thioesterase. (B) Left: mechanism for the cyclization of PMB3 catalyzed by the PMB TE domain. Right: proposed scheme for the cyclization of synthetic PMB3−SNAC substrate by purified PMB TE domain. (C) Purified PMB TE domain. MALDI-TOF mass spectrometry and (inset) SDS-PAGE analysis of the purified PMB TE domain.

polypeptides such as polymyxin B, surfactin, and fengycin.6,18,21,22 In the present study we functionally characterized the TE domain from polymyxin B (PMB) synthetase in order to gain a more detailed understanding of polymyxin biosynthesis, and gauge the potential of the isolated TE domain for semisynthetic applications in novel polymyxin discovery programs. The isolated PMB TE domain was demonstrated to catalyze the cyclization of a linear PMB-N-acetylcysteamine (SNAC) substrate analogue. Notably, the PMB TE contains an active site Cys as part of its catalytic triad as opposed to a Ser residue as found in most NRPS TE domains. Mutation of the native active site Cys to a Ser residue significantly reduced the catalytic efficiency of the PMB TE. Detailed examination of a homology model of the TE domain indicated that the substrate binding cleft is highly negatively charged, suggesting a novel

mechanism for interaction with the polycationic linear PMB peptidyl substrate.

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MATERIALS AND METHODS Chemical Reagents. Triisopropylsilane (TIPS), trifluoroacetic acid (TFA), and N-acetylcysteamine were purchased from Sigma-Aldrich (Sydney, New South Wales, Australia). Piperidine were obtained from Auspep (Melbourne, Victoria, Australia). Fmoc-Dab(Boc)-OH was purchased from Try-lead Chem (Hangzhou, Zhejiang, China). Fmoc-Thr(tBu)-OH and Fmoc-Leu-OH were from Mimotopes (Melbourne, Australia). Fmoc-Dab(ivDde)-OH, Fmoc-D-Phe-OH, and 1H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-chloro hexafluorophosphate-(1-),3-oxide (HCTU) were purchased from ChemImpex International (Wood Dale, Illinois, USA). FmocThr(tBu)-TCP-Resin was obtained from Intavis Bioanyltical Instruments (Köln, Cologne, Germany). Dimethylformamide

B


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Cloning of PMB TE. The synthetic PMB TE gene (GeneArt, ThermoFisher Scientific) was sequence optimized for expression in Escherichia coli, and to include a C-terminal HRV 3C protease recognition site and hexa-His tag. The synthetic gene was cloned into the BamHI and EcoRI restriction sites of the pRSET expression vector (ThermoFisher Scientific). Protein Expression and Purification. Tuner(DE3) pLysS competent cells (Merck Millipore) were transformed with the PMB TE expression plasmid. Cells were grown in LB media containing 50 μg/mL of ampicillin at 37 °C to an OD600 of 1.0 and then at 25 °C for 30 min. Protein expression was induced by the addition of IPTG to a final concentration of 0.2 mM, and the cells were incubated for an additional 18 h at 25 °C. The cultures were centrifuged at 3200g for 15 min at 4 °C to pellet the cells. The cells were resuspended in BugBuster (Merck Millipore) solution (10 mL/g of cells) containing 1× complete protease inhibitor cocktail (Sigma-Aldrich) and then lysed by sonication at 4 °C. The lysed cells were centrifuged at 48000g for 30 min at 4 °C, and the supernatant was loaded onto a 5 mL Ni2+-affinity HiTrap chelating column (GE Healthcare) equilibrated with buffer A (25 mM HEPES pH 8.0, 300 mM NaCl, 5 mM imidazole). Bound proteins were eluted with a linear gradient of 0−100% buffer B (25 mM HEPES pH 8.0, 300 mM NaCl, 250 mM imidazole). Fractions were analyzed by SDS-PAGE, and those containing hexa-His tagged PMB TE were pooled, exchanged into buffer A, and concentrated to 4 mg/mL. Hexa His-tagged HRV 3C protease (Life Technologies) was added at a ratio of 1U protease per 100 μg fusion protein, and the mixture was incubated at 4 °C for 48 h. The cleaved protein was passed through a 5 mL Ni2+affinity HiTrap chelating column to remove the cleaved hexaHis tag and hexa-His tagged protease. The eluate was exchanged into buffer C (25 mM HEPES pH 7.0, 50 mM NaCl) and concentrated. The purity and mass of purified PMB TE were determined by SDS-PAGE staining with Coomassie Brilliant Blue and MALDI-TOF mass spectrometry, respectively. Protein concentration was determined by measuring the absorbance at 280 nm and using a molar extinction coefficient of 27 515 M−1 cm−1.23 Site-Directed Mutagenesis. The Cys89Ser PMB TE mutant was generated by PCR using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies). Oligonucleotides used to generate the Cys89Ser mutant were designed according to the guidelines outlined in the QuikChange II XL site-directed mutagenesis kit and were synthesized by GeneWorks (Australia). The Cys89Ser mutation was confirmed by DNA sequencing. Enzymatic Assays. To 50 μL of 200 μM PMB3−S-NAC in buffer C was added 50 μL of 10 μM PMB TE. The reaction mix was incubated at 25 °C. Aliquots (30 μL) were taken at given time intervals, and the reaction was quenched by the addition of 3 μL of 1% TFA. The samples were immediately frozen with liquid nitrogen and stored at −20 °C. The samples were thawed, and to this was added 10 μL of acetonitrile before spinning at high speed for 5 min at 4 °C in a benchtop microfuge. The supernatant was analyzed by reversed-phase HPLC on a C18 using a linear gradient of 20−100% acetonitrile in 0.1% TFA over 30 min. NMR Data Acquisition and Processing. Samples of 0.7 mM PMB3−SNAC, the extracted PMB TE reaction products (above), or 1 mM PMB3 was dissolved in 50 mM deuterated sodium acetate buffer pH 4.5 containing 90% H2O/10% D2O

(DMF), methanol (MeOH), diethyl ether, dichloromethane (DCM), and acetonitrile were purchased from Merck (Melbourne, Victoria, Australia). Synthesis of PMB3 N-Acetylcysteamine Thioester. Synthesis of the Polymyxin B3 Protected Linear Peptide. Synthesis was conducted on a Protein Technologies Prelude automated peptide synthesizer using standard Fmoc solid-phase peptide chemistry. Specifically, synthesis was undertaken using TCP-Resin, preloaded with Fmoc-Thr(tBu)-OH (0.1 mmol scale). Coupling of the Fmoc-amino acids and the N-terminal octanoyl group was performed using the default instrument protocol: 3 mol equiv (relative to resin loading) of the Fmoc amino acid and HCTU in DMF with activation in situ, using 6 mol equiv of DIPEA. This was carried out for 50 min at room temperature. Fmoc deprotection was conducted using the default instrument protocol: 20% piperidine in DMF (1 × 5 min, 1 × 10 min) at room temperature. The protected linear peptide was then cleaved from the resin by treating the resin with 20% hexafluoroisopropanol (HFIP) in DCM (1 × 30 min, 1 × 5 min). This solution was concentrated in vacuo, and the resulting residue was taken up in 50% acetonitrile/water and freeze-dried for 2 days to give crude PMB3 protected linear peptide. Synthesis of the N-Acetyl Thioester. The crude PMB3 protected linear peptide (50 mg, 0.026 mmol) was dissolved in DMF (1 mL) to which HCTU (1.2 equiv, 0.031 mmol, 13 mg), DIPEA (3 equiv, 0.078 mmol, 14 μL), and Nacetylcysteamine (2.5 equiv, 0.065 mmol, 7 μL) were added. The solution was stirred at room temperature for 30 min and then concentrated in vacuo. The resulting residue was taken up in a solution 5%TIPS/TFA (5 mL) containing 100 μL of Nacetylcysteamine (which acts as a thiol based scavenger), and the solution was left to agitate on a shaker table for 1.5 h. The TFA was evaporated under a gentle stream of nitrogen gas, and diethyl ether (40 mL) was added to the remaining residue. The resulting precipitate was collected then air-dried in a fume hood to give the crude PMB3 N-acetylcysteamine thioester as a white solid. The resulting solid was taken up in 0.1% TFA/Water (5 mL) and subjected to RP-HPLC purification. This was carried out on a Waters Prep LC system connected to a Waters 486 tunable absorbance detector (214 nm). A Phenomenex Axia column (Luna C8(2), 250 × 21.2 mm ID, 100 Å, 10 μm) was employed with a gradient of 0−60% solvent B over 60 min at a flow rate of 15 mL/min; solvent A was 0.1% TFA/water and solvent B was 0.1% TFA/acetonitrile. Collected fractions were analyzed using a Shimadzu 2020 LCMS system, incorporating a photodiode array detector (214 nm) coupled directly to an electrospray ionization source and a single quadrupole mass analyzer. RP-HPLC was carried out with a Phenomenex column (Luna C8(2), 100 × 2.0 mm ID) eluting with a gradient of 0−60% solvent B over 10 min at 0.2 mL/min; solvent A was 0.05% TFA/water, and solvent B was 0.05% TFA/acetonitrile. Mass spectra were acquired in the positive ion mode with a scan range of 200−2000 m/z. Fractions containing the desired product were freeze-dried for 2 days to give the PMB3 N-acetylcysteamine thioester TFA salt as a white solid in a yield of 15.0 mg. The purity was 98.5% as determined by RP-HPLC at 214 nm. The compound was confirmed as having the correct molecular weight by ESI-MS analysis: m/z (monoisotopic) calculated; C59H105N17O14S [M + H]+ 1308.8, [M + 2H]2+654.9, [M + 3H]3+ 436.9; observed; [M + H]+ 1309.2, [M + 2H]2+ 655.5, [M + 3H]3+ 437.3. C


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1188.8 Da, which corresponds closely to the theoretical monoisotopic mass of cyclic PMB3 (1188.7) (Figure 2A−D).

and 0.02% (w/v) sodium azide. NMR experiments were performed at 283 or 298 K on a Bruker AVANCE NMR spectrometer operating at a 1H frequency of 600 MHz and equipped with a cryogenically cooled triple-resonance TXI probe. Backbone resonance assignments were obtained using the following two-dimensional (2D) experiments: 1H−15N HSQC, 1H−1H COSY, 1H−1H TOCSY (mixing time 30 and 80 ms) and 1H−1H NOESY (mixing time 80 and 200 ms). Water suppression in homonuclear experiments was achieved using a 3-19-19 WATERGATE sequence, and gradients were used to select coherences in the heteronuclear experiments.24 Chemical shifts were referenced to 2,2-dimethyl-2-silapentane5-sulfonic acid (DSS). NMR data were processed using NMRPipe or the Bruker software TopSpin and analyzed using NMRView.25 Chemical shifts changes (Δδ) were calculated using eq 1: Δ=

(Δδ H)2 + (c Δδ N)2

(1)

where ΔδH and ΔδN are the chemical shift changes of amide proton and nitrogen, respectively, and c is a scaling factor. Differential Scanning Fluorometry. Thermal melt analyses under varying buffer and pH conditions (Buffer Screen 9)26 were performed on the PMB TE protein and analyzed using the program Meltdown27 (C3 Facility, CSIRO, Australia). Briefly, the protein was diluted into the test solution in 96-well PCR plate in a total volume of 20 μL. Each sample contained 0.3 μL of 1 mg/mL protein, 0.3 μL of a 1:10 dilution of Sypro dye in water, and 19.4 μL of buffer solution containing 50 or 200 mM NaCl at the desired pH. Assays were performed using a BioRad CFX96 RT-PCR machine varying the temperature from 20 to 100 °C at 0.5 °C per 0.5 min. Homology Modeling. In silico modeling of PMB TE was performed with the structure prediction programs MODELER28 and HHpred,29 and protein structures were visualized and superimposed with PyMOL.30

Figure 2. PMB thioesterase domain catalyzed cyclization of PMB3− SNAC. (A) HPLC profiles and (B) progress curve for reactions containing 100 μM PMB3−SNAC and 6 μM PMB TE in 25 mM HEPES buffer containing 50 mM NaCl (pH 7.0, 25 °C). (C, D) LCMS analysis of (C) the synthetic PMB3−SNAC substrate and (D) cyclized product (PMB3) formed by the PMB TE catalyzed cyclization of PMB3−SNAC. (E) Sum of the HPLC peak area for the substrate (PMB3−SNAC) and product (PMB3) for the PMB TE domain catalyzed cyclization of PMB3−SNAC over time. (F) Stability of the PMB TE domain at 4 (red bars) and 25 °C (blue bars). The PMB TE domain was incubated at either 4 or 25 °C for 30 or 60 min and then added to a reaction mixture containing 6 μM PMB TE, 100 μM PMB3−SNAC in 25 mM HEPES pH 7.0 and 50 mM NaCl. The reaction mixture was incubated at 25 °C, and samples were taken at various time intervals and analyzed by reverse-phase HPLC.

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RESULTS Overexpression and Purification of the Polymyxin B Synthetase TE Domain. PMB TE was expressed in E. coli Tuner(DE3) pLysS and purified by nickel affinity chromatography as outlined in the experimental procedures. SDS-PAGE analysis showed that the purified protein was greater than 95% pure, and MALDI mass spectrometry analysis gave an experimental mass of 30 010 Da, which was close to the theoretical value of 29 201 Da (Figure 1C). TE Catalyzed Cyclization of a Linear Polymyxin B−NAcetylcysteamine (PMB3−SNAC) Substrate Analogue. The S-N-acetylcysteamine (SNAC) derivative of linear polymyxin B3 (PMB3−SNAC, octanoyl-Dab-Thr-Dab-DabDab-D-Phe-Leu-Dab-Dab-Thr-SNAC (linear PMB3−SNAC), (Figure 1B) was utilized to examine the ability of TE to catalyze the final cyclization step in the polymyxin NRPS to form cyclic polymyxin B3 (PMB3). N-Acetylcysteamine is structurally identical to the phophopantetheine moiety found in the native peptidyl carrier protein (PCP) domain and is therefore a good mimic for the natural substrate PMB3-SPCP.18,28,31 To determine whether the PMB TE can catalyze the cyclization of PMB3−SNAC, we incubated a solution containing the purified PMB TE with the PMB3−SNAC substrate at 25 °C and monitored the reaction by LC-MS analysis. The results showed that recombinant TE cyclized PMB3−SNAC to form a product with a monoisotopic mass of

In the heat denatured PMB TE control experiment, no conversion to the cyclic form was detected, which essentially demonstrates only the active enzyme can catalyze substrate cyclization (data not shown). NMR spectroscopy was employed to examine the product formed by the cyclization of PMB3−SNAC. Peaks in the 1H−15N HSQC spectrum of the substrate PMB3−SNAC were assigned by the standard sequential assignment procedure using 2D homonuclear NOSEY, ROESY, TOCSY and COSY NMR spectra (Table S1 and SI Figure 1). The lack of long-range NOEs in the 1 H−1H ROESY spectrum indicated that linear PMB3−SNAC was disordered in solution and did not adopt a predefined conformation prior to binding to the TE active site (SI Figure 1B). Chemical shifts of resonance peaks in the 1H−15N HSQC NMR spectrum for the cyclic product were identical to those of the fully synthetic pure PMB3 (Figure 3). The peak corresponding to Dab9 was not observed consistent with our D


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Stability of Polymyxin B TE. To test the stability of the PMB TE, we incubated the protein at 4 or 25 °C for several hours prior to the addition of substrate. The amount of product formed with time was similar for preincubation of the enzyme at either temperature showing that the enzyme is stable under the conditions used in the assay (Figure 2F). We used MALDITOF to determine whether a reaction product was inhibiting the PMB TE. MALDI analysis of PMB TE in the presence of the substrate showed the formation of a higher molecular weight species with a mass approximately 645 Da larger than TE (SI Figure 2). We also examined the ability of the PMB TE to catalyze the cyclization of PMB3−SNAC at various pH values. Purified PMB TE was buffer exchanged into various buffers at pH 4.5, 6.0, or 7.0 and then incubated with the substrate PMB3−SNAC prepared in the same buffer. Both hexa-His tagged and untagged PMB TE exhibited reduced activity at pH 6.0 and were inactive at pH 4.5 (SI Figure 3A,B). It is possible that this decrease in activity at lower pH values may be due to inhibition of catalysis (e.g., ionization of a catalytic important residue) or instability of the protein. Differential scanning fluorimetry (DSF) was used to determine whether this loss in activity was due to the instability of the protein at lower pH values. The DSF data showed that PMB TE is unstable at pH 5.0, which is not unexpected since it is close to the predicted pI values for hexa-His tagged and untagged PMB TE (4.76 and 4.59, respectively) (SI Figure 3C). In contrast, the PMB TE exhibited a similar degree of stability between pH 6.0 and 7.0, even though its activity was significantly reduced at pH 6.0. This would suggest that a catalytically important residue may undergo a change in ionization state upon going from pH 7.0 to 6.0 resulting in a loss of activity The Polymyxin B TE Domain Employs a Unique Catalytic Cysteine. Alignment of the amino acid sequences of polymyxins with other NRPs thioesterases reveals that the Ser residue normally found in the catalytic triad of most NRPS TE domains is replaced by a Cys residue in position 89 of the PMB TE (Figure 4A). We examined the effect of different reducing agents on the catalytic activity of the enzyme and substrate stability. Interestingly, incubation of the PMB3−SNAC/PMB TE reaction mixture in the presence of the reducing agent DTT caused substrate to rapidly decompose (SI Figure 4), whereas incubation with TCEP did not impact substrate stability. To determine whether the mutation of the catalytic Cys to a Ser residue affected the activity of the enzyme, we generated the Cys89Ser mutant (Figure 4B). We compared that ability of the Cys89Ser mutant and wild-type PMB TE to catalyze the cyclization of the PMB3−SNAC substrate. The Cys89Ser mutant exhibited an approximately 60-fold lower activity (based on the initial rates) than the wild type enzyme indicating that the catalytic Cys residue was required for optimal activity (Figure 4B). Homology Model of Polymyxin B TE. Different strains of Paenibacillus polymyxa contain polymyxin NRPSs which produce specific types of polymyxins, such as polymyxins A, B, E, and P.33−36 Polymyxin synthetases contain TE domains which display a high degree of sequence homology (SI Figure 5). In contrast, the amino acid sequences for different TEs are quite variable (SI Figure 6A), while the residues that are conserved lie predominantly within the core of the proteins (SI Figure 6B,C). The structures for a number of TEs have been determined, and these proteins display a reasonably high degree

Figure 3. NMR analysis of the product formed from the cyclization of PMB3−SNAC by PMB TE. Comparison of the 1H−15N HSQC spectra for (A) The substrate PMB3−SNAC (red) and the PMB3 product formed in the presence of the PMB TE (blue). (B) Synthetic PMB3 standard (green) and the PMB3 product formed in the presence of the PMB TE (blue). (C) Synthetic PMB3 standard (red); the substrate PMB3−SNAC (blue) and the PMB3 product formed in the presence of the PMB TE (cyan). The yellow arrow indicates the amide resonance from the SNAC.

previous NMR studies of a dansyl-derivative of PMB3.32 Additional peaks were also observed corresponding to the substrate PMB3−SNAC which was not unexpected since some unutilized substrate usually remained in the reaction mixture. Notably, residues Phe6 to Thr10 of the linear PMB3−SNAC substrate exhibited the largest chemical shift perturbations upon transition to cyclic PMB3, owing to changes in the chemical environment of their respective backbone amide protons (Figure 3 and SI Figure 1C). Residue Thr10 which forms a covalent bond with Dab4 displayed the largest change in chemical shift (SI Figure 1C). Overall, the NMR results confirmed that the product has a structure identical to that of PMB3. The progress curve for the cyclization of PMB3−SNAC by PMB TE shows that the reaction did not go to completion (Figure 2B). This suggests that the PMB TE might be unstable under the assay conditions or a product of the reaction inhibited the enzymes activity. E


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similarity allowed us to generate a high fidelity homology model of the PMB TE domain (Figure 6A). The overall structure of the homology model of the PMB TE domain is most similar to the X-ray crystal structure for the surfactin TE domain from Bacillus subtilus, while the model deviates from that of fengycin TE within the lid region (Figure 6A). The PMB TE model structure consists of a fold similar to that of α/ β hydrolases and a bowl-shaped active site cavity. The Cys-HisAsp catalytic triad composed of residues Cys89, His218, and Asp116 (numbered from the N-terminus of the PMB TE domain) adopts a conformation similar to the Ser-His-Asp triad found in the majority of other TE domains (Figure 6A). Poly TE Substrate Binding Cleft Is Highly Charged. We examined the PMB TE homology model to gain further detailed insights into the mechanism of action of this enzyme. Analysis of the surface electrostatic potential of PMB TE shows that the substrate binding pocket is highly negatively charged (Figure 6B), while those for fengycin and surfactin TEs are predominately neutral except for the region comprising the catalytic triad (Figure 6C,D). It is therefore likely that, unlike fengycin and surfactin TEs, charged interactions play a predominate role in the binding of the positively charged substrate (linear PMB) to the negatively charged PMB TE binding site.

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DISCUSSION The enormous structural and functional diversity of secondary metabolite cyclic peptides, such as the polymyxins, is attributable to their nonribosomal mode of biosynthesis by large multifunctional enzymes termed NRPS. We and several other groups have sequenced and characterized pmx biosynthetic gene clusters from a number of P. polymyxa strains (polymyxin A,33 polymyxin B,35 polymyxin D (our unpublished results), polymyxin E,36 polymyxin P34), which offers new opportunities to design the production of novel polymyxins by genetically engineering the biosynthetic pathway in a surrogate host. The pmx gene cluster consists of five open reading frames, pmxA, pmxB, pmxC, pmxD, and pmxE (Figure 1A).35 Three of the genes pmxA, pmxB and pmxE are directly involved in polymyxin synthesis, while pmxC and pmxD encode selfresistance ABC transporters responsible for export of the lipopeptide out of the cell. The pmxA (15 kb) gene encodes for a synthetase of 4,998 amino acids (aa), pmxB (3.3 kb) a synthetase of 1,102 aa and pmxE (18.9 kb) a synthetase of 6,292 aa. The three NRPSs (PmxA, PmxB, and PmxE) contain different numbers of modules, PmxB contains a single module while PmxA and PmxE are comprised of four and five modules, respectively. Each module is comprised of condenstation, adenylation and peptide carrier protein (C-A-PCP) domains while module 6 of PmxA harbors an additional auxiliary epimerization domain. This epimerization domain catalyzes the generation of a D-configured amino acid from the corresponding L-isomer when attached to the 4′-Phosphopantetheine “swinging arm” cofactor of the cognate PCP.45 The PmxA, PmxB and PmxE synthases combine to form an ordered assembly line for the synthesis of the lipopeptide. During the synthesis of nonribosomal peptides, the growing peptide-chain is transferred from one module to the next, such that the growth of the peptide occurs by an ordered succession of transpeptidation and condensation reactions (Figure 1A). The terminal step of PMB assembly results in the release of the

Figure 4. PMB TE domain. (A) Alignment of the amino acid sequence for the TE domains of polymyxins A, B, E, and P with those of fengycin and surfactin. Active site residues are colored, red while those comprising the lid region and β7-αE loop are highlighted in yellow and gray, respectively. Secondary structural motifs derived from the X-ray crystal structures of fengycin and surfactin are shown above the sequence alignment where α-helices and β-strands are represented as red cylinders and blue arrows, respectively. (B) Left panel: SDSPAGE analysis of (lane 1) purified wild type PMB TE and (lane 2) the C89S-PMB TE mutant. Right panel: progress curves for the cyclization of PMB3−SNAC by wild type PMB TE (blue squares) and the catalytic site mutant C89S-PMB TE (red dots). The reaction mixture contained 50 μM enzyme and 100 μM PMB3−SNAC in 25 mM HEPES pH 7.0, 50 mM NaCl. Reactions were incubated at 25 °C, and samples taken at given time intervals were analyzed by reverse-phase HPLC.

of structural homology despite their low sequence similarity (SI Figure 6D).12,37−42 In all cases, these TEs contain a highly conserved His-Asp-Ser catalytic triad,12,43 which is commonly found among the α-/β-hydrolase-fold family of enzymes.44 Polymyxin TE domains exhibit a significant degree of sequence homology to the TE domains of fengycin (67% conservation) and surfactin (63% conservation) for which X-ray crystallographic structures are available (Figure 4A, SI Table 2).37,38 Analysis of the PMB TE amino acid sequence using a number of secondary structural prediction programs also indicates that it possesses a structure similar to that of the fengycin and surfactin TE domains (SI Figure 5). This F


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Figure 5. PMB TE possesses a secondary structure similar to that of fengycin and surfactin thioesterases. Predicted secondary structural motifs of PMB TE determined using the prediction programs Jpred 4,67 SSPred,68 SSPro8,69 PsiPred,70 REPROFsec,71 NetSurfP,72 and Porter.73 β-Strands are represented by an E, α-helices by H, and loops by C. Secondary structural motifs taken from the crystal structures of fengycin (PDB ID: 2CB9) and surfactin (PDB ID: 1JMK). TEs are shown above the polymyxin B amino acid sequence where α-helices are represented as cylinders, β-strands as arrows. α-Helices comprising the lid region of fengycin and surfactin TEs are colored green.

Examination of the TE family of enzymes within the Pfam database52 indicated that less than 2% of all TEs contain a catalytic Cys residue. Comparison of the sequence alignments of several TEs containing a catalytic Cys, as opposed to those with a catalytic Ser, shows that most residues comprising the catalytic triad are conserved; however the Asp residue is replaced by a Ser or Asn in a significant number of cases (SI Figures 7 and 8). In contrast, this Asp residue is largely conserved in proteins containing the Ser-His-Asp catalytic triad. The Asp in the Ser-His-Asp catalytic triad forms a hydrogen bond with the His residue increasing the pKa of its imidazole nitrogen.53 This allows the His residue to act as a powerful general base and to activate the Ser nucleophile. However, due to the intrinsically lower pKa of Cys, the Asp residue may play a less important role in the Cys-His-Asp catalytic triad. The catalytic Cys is located at the N-terminus of an α-helix segment, and it is known that dipole effects can lower the pKa of the catalytic Cys,54−56 as has been reported for various Cys proteases.54 Further evidence for the role of the Cys in the Cys-His-Asp catalytic triad comes from studies of another α/β-fold hydrolase family member, dienelactone hydrolase (DLH).44 DLH possesses a Cys-His-Asp catalytic triad similar to PMB TE and is an enzyme used by bacteria and fungi to degrade aromatic compounds such as dienelactone to maleylacetate. It has been reported that substitution of the catalytic Cys of DLH with Ser produced an enzyme with only 10−15% of the wild type activity.57 The proposed model explaining why the Cys nucleophile in the triad of DHL that is catalytically competent involves substrate-assisted catalysis, where a functional group in the substrate plays a key role in the catalytic mechanism.58−60 It has been proposed that the carboxylate group of the dienelactone substrate acts as a molecular switch that controls the protonation state of the catalytic Cys nucleophile. In the

mature peptide by ring closure catalyzed by the TE domain situated at the C-terminus of PmxB (i.e., module 10). We have shown that the isolated TE domain of the PMB NRPS can catalyze the cyclization of a linear octanyl-PMB3− SNAC analogue to form the mature lipopeptide PMB3. This would suggest the presence of the N-terminal octanyl fatty acyl does not interfere with the cyclization of the linear PMB. As there is no precedent for this mechanism for the polymyxins, this opens up the possibility that the N-terminal addition of the octanyl fatty acyl occurs, while the nascent linear PMB peptidyl intermediate is tethered to the NRPSs. Since the PMB NRPSs cluster does not contain a fatty acyl transferase domain, this must be an axillary modification that is performed by an as yet to be discovered specific PMB fatty acyl ligase enzyme.33 The polymyxin TE domains deviate from the canonical SerHis-Asp catalytic triad possessed by the majority of NRPS TE and contain a catalytic Cys residue instead of Ser in the catalytic triad. This is supported by the fact that PMB TE is completely inactivated by reducing agents. Other TEs that contain a GXCXG rather than the usual GXSXG catalytic motif include the pyochelin NRPS type I TEs from Pseudomonas aeruginosa46 and Streptomyces venezuelae47 and the penyloxazoline NRPS type I TE from Mycobacterium tuberculosis.48 Several type II thioesterases also contain a catalytic Cys residue including those from Catenulispora acidiphila and Ruminococcus albus. It has previously been reported that modification of the catalytic Ser of type I thioesterases to a Cys residue results in a significant decrease in enzymatic activity,17,49 while mutating the catalytic Ser residue of several type II thioesterases to Cys produces an enzyme with only marginally reduced activity.50,51 Similarly, mutation of the catalytic Cys of PMB TE to a Ser residue resulted in a 60-fold reduction in enzymatic activity confirming that this residue is essential for catalysis (Figure 4B). G


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Biochemistry

Figure 6. Comparison of the PMB, fengycin, and surfactin TE domains. (A) Left panel - homology model of the PMB TE domain where regions known to be important for catalysis and substrate specificity are colored orange (lip region) and pink (β7-αE loop). Right panel - comparison of the PMB TE homology model (green) generated using the 3D structure prediction program HHPred with the X-ray crystal structures of fengycin (blue; PDB ID: 2CB9) and surfactin (magenta; PDB ID: 1JMK). Residues comprising the catalytic triad are represented as sticks (purple). (B−D) Surface electrostatic potential for the (left panel) enzyme and (right panel) substrate for (B) PMB TE, (C) fengycin, and (D) surfactin. Residues belonging to the catalytic triad are labeled. Positively charged regions are colored blue, neutral regions are white, and negatively charged regions are red. Models of linear PMB3 and cyclized PMB3, fengycin and surfactin were built and energy minimized in ChemDraw (https://www.cambridgesoft.com/ software/overview.aspx), while the surface electrostatic potential was generated in MAESTRO (http://www.schrodinger.com/).

the His residue, but rather forms a hydrogen bond with a Glu residue that is in close proximity, and in the absence of substrate this Glu residue is ion paired with an Arg. Upon

free state, the His and Asp residues of the catalytic triad form an ion pair, while the Cys exists as a thiol that is not catalytically competent. The thiol group of the Cys does not interact with H


Article

Biochemistry

Figure 7. Proposed mechanism for the PMB TE catalyzed cyclization of PMB3−SNAC. Positively charged residues within the N-terminal region of PMB3−SNAC are attracted to the surface of the PMB TE in close proximity to Thr10. The PMB TE catalyzed generation of a covalent bond between the side chain amino group of Dab4 and backbone carbonyl of Thr10 results in the formation of cyclic PMB3. Positively charged Dab resides are denoted by red plus signs and are highlighted by light-blue ellipses. The negatively charged substrate-binding site of PMB TE is colored red and denoted by blue minus signs.

place while the nascent peptide is still bound to the NRPS. Essentially, the prefolded conformation places the terminal residues in close spatial proximity within the catalytic site of the TE domain, thereby facilitating efficient formation of a covalent bond leading to the cyclized peptide. In contrast, our NMR studies indicate the linear PMB3 substrate does not adopt a transient prefolded structure but is entirely disordered in solution. The lack of intrinsic prefolding might be explained by the highly negatively charged substrate binding cleft observed in the homology model of the PMB TE domain (Figure 6B). It is more likely that electrostatic interactions between the highly positively charged PMB and negatively charged residues within the TE binding cleft direct the folding of PMB prior to cyclization (Figure 7). Owing to their modular structure, NRPSs are highly amendable to genetic level combinatorial approaches for the rational design of custom NRPSs that are capable of expressing novel polymyxin lipopeptides and daptomycin analogues in a surrogate host.6,8,63,64 Therefore, incorporating custom designed NRPSs into biosynthetic platforms has the potential to enable the fermentative production of novel polymyxins on an industrial scale. Toward this goal, we have undertaken a detailed functional characterization of the isolated PMB TE domain. The TE-domains of NRPS only display selectivity for terminal amino acids of the linear peptide chain that form a covalent bond (i.e., the electrophilic and the nucleophilic amino acids) leading to the cyclic peptide; in the case of the naturally occurring polymyxins these are invariably Thr and Dab. This is very advantageous as the internal sequence of the cyclic peptide

substrate binding, this Arg binds to a carboxylate group of the substrate, the Glu-His bond is weakened and the His moves toward the Cys and abstracts a hydrogen atom from its thiol group. The ionized Cys residue can then form a stabilizing bond with the His of the catalytic triad. In this way the thiolate group is only generated in the presence of substrate. It is tenable to imagine a similar mechanism may be at play with the catalytic mechanism of the PMB TE. TE domains catalyze product release by a two-step reaction mechanism: (1) Transfer of the full-length peptide chain attached to the terminal PCP to a highly conserved Ser (or Cys) residue in the active site of the TE-domain via the formation of an acyl-O-TE intermediate; and (2) subsequent cleavage and release by a regio- and stereoselective intramolecular macrocyclization reaction using a peptide internal nucleophile to produce a cyclic peptide (e.g., daptomycin and polymyxins)19,45 or attack by a water molecule to yield a linear peptide (e.g., vancomycin).19,20 Interestingly, we observed that the PMB3−SNAC cyclization reaction did not go to completion, suggesting that a reaction product may inhibit the isolated PMB TE. This was supported by the identification of a species in the reaction mixture with a mass approximately 645 Da larger than PMB TE. We suspect that the 645 kDa adduct is a degradation fragment of the PMB3−SNAC decapeptide that has stalled the PMB TE. A few reports have indicated that intramolecular hydrogen bonding in certain nonribosomal peptides allows them to adopt a transiently stable conformation in solution that resembles the mature cyclic product.61,62 This prefolding is believed to take I


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(3) Velkov, T., Roberts, K. D., Thompson, P. E., and Li, J. (2016) Polymyxins: a new hope in combating Gram-negative superbugs? Future Med. Chem. 8, 1017−1025. (4) Velkov, T., Thompson, P. E., Nation, R. L., and Li, J. (2010) Structure-activity relationships of polymyxin antibiotics. J. Med. Chem. 53, 1898−1916. (5) Cochrane, S. A., and Vederas, J. C. (2016) Lipopeptides from Bacillus and Paenibacillus spp.: A gold mine of antibiotic candidates. Med. Res. Rev. 36, 4−31. (6) Baltz, R. H. (2014) Combinatorial biosynthesis of cyclic lipopeptide antibiotics: a model for synthetic biology to accelerate the evolution of secondary metabolite biosynthetic pathways. ACS Synth. Biol. 3, 748−758. (7) Meier, J. L., and Burkart, M. D. (2009) The chemical biology of modular biosynthetic enzymes. Chem. Soc. Rev. 38, 2012−2045. (8) Walsh, C. T. (2004) Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805−1810. (9) 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. (10) Konz, D., and Marahiel, M. A. (1999) How do peptide synthetases generate structural diversity? Chem. Biol. 6, R39−R48. (11) Strieker, M., Tanović, A., and Marahiel, M. A. (2010) Nonribosomal peptide synthetases: structures and dynamics. Curr. Opin. Struct. Biol. 20, 234−240. (12) Marahiel, M. A. (2016) A structural model for multimodular NRPS assembly lines. Nat. Prod. Rep. 33, 136−140. (13) Weber, T., and Marahiel, M. A. (2001) Exploring the domain structure of modular nonribosomal peptide synthetases. Structure 9, R3−R9. (14) Koglin, A., Löhr, F., Bernhard, F., Rogov, V. V., Frueh, D. P., Strieter, E. R., Mofid, M. R., Güntert, P., Wagner, G., Walsh, C. T., Marahiel, M. A., and Dötsch, V. (2008) Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 454, 907−911. (15) Doekel, S., and Marahiel, M. A. (2001) Biosynthesis of natural products on modular peptide synthetases. Metab. Eng. 3, 64−77. (16) Marahiel, M. A., and Essen, L. O. O. (2009) Chapter 13. Nonribosomal peptide synthetases mechanistic and structural aspects of essential domains. Methods Enzymol. 458, 337−351. (17) Shaw-Reid, C. A., Kelleher, N. L., Losey, H. C., Gehring, A. M., Berg, C., and Walsh, C. T. (1999) Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization. Chem. Biol. 6, 385−400. (18) Keating, T. A., Ehmann, D. E., Kohli, R. M., Marshall, C. G., Trauger, J. W., and Walsh, C. T. (2001) Chain termination steps in nonribosomal peptide synthetase assembly lines: directed acyl-Senzyme breakdown in antibiotic and siderophore biosynthesis. ChemBioChem 2, 99−107. (19) Walsh, C. T. (2016) Insights into the chemical logic and enzymatic machinery of NRPS assembly lines. Nat. Prod. Rep. 33, 127−135. (20) Grünewald, J., and Marahiel, M. A. (2006) Chemoenzymatic and template-directed synthesis of bioactive macrocyclic peptides. Microbiol. Mol. Biol. Rev. 70, 121−146. (21) Kohli, R. M., Trauger, J. W., Schwarzer, D., Marahiel, M. A., and Walsh, C. T. (2001) Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases. Biochemistry 40, 7099−7108. (22) Du, L., and Lou, L. (2010) PKS and NRPS release mechanisms. Nat. Prod. Rep. 27, 255−278. (23) Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J. C., Williams, K. L., Appel, R. D., and Hochstrasser, D. F. (1998) Protein identification and analysis tools in the ExPASy server. Methods Mol. Biol. 112, 531−552. (24) Piotto, M., Saudek, V., and Sklenár,̌ V. (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661−665.

can be varied without affecting the peptide cyclization reaction. Furthermore, there has been increased interest in the semisynthesis of cyclic peptides utilizing chemoenzymatic routes. Enzymatically catalyzed cyclization reactions are much faster than standard chemical cyclization methodology and can be executed under aqueous conditions, which improves efficiency and safety.65,66 Therefore, the combination of powerful solid phase peptide synthesis, that allows the incorporation of diverse monomer building blocks into linear peptidyl substrates, and regio- and stereoselective enzymatic peptide cyclization utilizing the TE-domain, could be very useful in the synthesis of non-natural polymyxin lipopeptides. To this end we are currently exploring the potential of this chemoezymatic methodology in the preparation of novel polymyxins in our laboratory, with particular focus on increasing the efficiency of the TE catalyzed cyclization reaction.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b01139. Figure S1. Chemical shift assignments for PMB3−SNAC. Figure S2. MALDI-TOF data for the PMB TE. Figure S3. pH and thermal stability data for the PMB TE. Figure S4. Impact of reducing agents on the PMB TE. Figure S5. Sequence alignment of polymyxin synthetase TE domains. Figure S6. Sequence and structural comparisons of the PMB TE with related NRPS TE domains. Figure S7. Sequence alignment of NRPS domains with catalytic Cys residues. Figure S8. Sequence alignment of NRPS domains with catalytic Ser residues (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(T.V.) E-mail: [email protected]. Telephone: +61 3 99039539. *(J.L.) E-mail: [email protected]. ORCID

Charles A. Galea: 0000-0003-2730-1105 Tony Velkov: 0000-0002-0017-7952 Funding

This work was supported by Australian National Health and Medical Research Council Grant (1064896). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.L. is an Australian National Health and Medical Research Council Senior Research Fellow. T.V. is an Australian National Health and Medical Research Council Industry Career Development Research Fellow.

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REFERENCES

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Functional Characterization of the Unique Terminal Thioesterase

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