Thioesterase-Mediated Synthesis of Teixobactin Analogues

Subscriber access provided by READING UNIV

Note

Thioesterase-mediated synthesis of teixobactin analogues: mechanism and substrate specificity Dhanaraju Mandalapu, Xinjian Ji, Jinfeng Chen, Chuchu Guo, Wan-Qiu Liu, Wei Ding, Jiahai Zhou, and Qi Zhang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02462 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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.

The Journal of Organic Chemistry 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 15 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 Journal of Organic Chemistry

Thioesterase-mediated synthesis of teixobactin analogues: mechanism and substrate specificity Dhanaraju Mandalapu,1 Xinjian Ji,1 Jinfeng Chen,2 Chuchu Guo,1 Wan-Qiu Liu,1 Wei Ding,1 Jiahai Zhou,2 and Qi Zhang1* 1

Department of Chemistry, Fudan University, Shanghai 200438, China

2

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of

Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China *

To whom correspondence should be addressed: 2005 Songhu Road, Fudan University, Chemistry

Building, Room 5019, Shanghai, 200438, China. Email: [email protected]

Abstract A chemoenzymatic approach for the synthesis of teixobactin analogues has been established by using the tandem thioesterase (TE) of the non-ribosomal peptide synthase (NRPS) Txo2. We show that, unlike the closely-related counterparts involved in lysobactin biosynthesis (in which the N-terminal TE is solely responsible for the lactonization reaction), the two teixobactin TE domains are functionally exchangeable and likely act synergistically, representing an unprecedented off-loading mechanism in NRPS enzymology. The substrate specificity of this tandem TE was also investigated in this study.

Teixobactin (Figure 1A) is a depsipeptide isolated from a β-proteobacterium Eleftheria terrae by using a sophisticated culturing method named iChip.1 This compound shows excellent antimicrobial

activity

against

many

methicillin-resistant

Staphylococcus

vancomycin-resistant

Enterococcus,

notorious aureus,

Gram-positive

pathogens,

vancomycin-intermediate

penicillin-resistant

Streptococcus

S.

including aureus,

pneumonia,

and

Mycobacterium tuberculosis.1 Teixobactin binds both lipid II and lipid III, thereby inhibiting the biosynthesis of peptidoglycan and wall teichoic acid (WTA).1-4 Thus far no teixobactin-resistant bacterial strains were isolated despite of extensive efforts, raising a great promise for the emergence of future antibiotics to fight against antimicrobial resistance (AMR).1 As a result, teixobactin has received widespread coverage in the international press,5-8 and its structure activity relationship has been extensively investigated by many groups of scientists in the past three years.4,9-22 1

ACS Paragon Plus Environment

The Journal of Organic Chemistry 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 2 of 15

The biosynthetic gene cluster of teixobactin was identified by an in silico analysis of the sequenced genome, which mainly consists of two large non-ribosomal peptide synthetase (NRPS) genes (txo1 and txo2) consisting of eleven modules in total (Figure 1B, a proposed biosynthetic pathway of teixobactin is also shown in Figure S1).1 However, no genetic or biochemical evidence for teixobactin biosynthetic gene cluster has been reported thus far. In this Note, we report functional dissection of the thioesterase (TE) domains in the teixobactin NRPS assembly line. This study not only validates the teixobactin gene cluster but more importantly, reveals an unusual off-loading mechanism in NRPS enzymology and sets up a chemoenzymatic approach for the synthesis of teixobactin analogues.

Figure 1. Teixobactin and its biosynthesis. (A) Chemical structure of teixobactin. D-amino acids are shown in red, and the unusual L-allo-enduracididine residue is shown in blue. (B) The gene and domain organization of Txo1 and Txo2, two NRPSs involved in teixobactin biosynthesis. The amino acid residues incorporated in each module (blue bar) are shown, and the tandem TE domains are shown in magenta. A proposed biosynthetic pathway of teixobactin is shown in Figure S1

Most of NRPSs and polyketide synthases (PKSs) contain a TE domain in the C-termini for product release. These TE domains contain a Ser residue that acts as catalytic nucleophile to 2


Page 3 of 15 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


produce a covalent acyl-O-Ser-TE intermediate from an acylthioester substrate, which is then released from the enzyme via a hydrolysis or cyclization step.23 The Txo2 termination module that contains two TE domains (TE1-TE2) (Figure 1B) was expressed in Escherichia coli with an N-terminal hexa-histidine tag, and the protein was purified by Ni2+ affinity chromatograph. Static light scattering (SLS) analysis showed that TE1-TE2 is a monomer in solution (Figure S2). To study the in vitro activity of TE1-TE2, we synthesized a peptide C-terminal methyl ester 1, whose sequence is consistent with that of teixobactin (except for L-Phe1 and L-Lys10, which substitute the N-Me-Phe1 and the unusual L-allo-enduracididine residue of teixobactin, respectively) (Table 1). We chose the methyl ester because it is much easier to be chemically synthesized than the corresponding N-acetylcysteamine (SNAC) thioester, the latter has been commonly used in TE-mediated in vitro reaction.24-28 Liquid chromatography (LC) with high resolution mass spectrometry (HRMS) analysis of the reaction mixture containing 1 and TE1-TE2 showed production of a compound exhibiting a protonated molecular ion at m/z = 601.8607 (z=2) (Figure 2, trace i), which was absent in the control assay with the supernatant of boiled enzyme (Figure 2, trace ii). The suggested molecular ion (C57H97N13O15, 1.0 ppm error) is consistent with the teixobactin analogue P1, and this analysis was further supported by detailed HR-MS/MS analysis (Figure S3). In addition to P1, we also observed the hydrolyzed product, which is ～3 fold higher in intensity than P1 (Figure S4); similar observations were common in TE in vitro reactions for cyclic peptide synthesis.24-28

Figure 2. LC-MS analysis of the reactions catalyzed by teixobactin TEs, showing the extracted ion chromatograms (EICs) of [M + H]2+ = 601.9 (corresponding to P1) for the reactions with (i) the wild type TE1-TE2, (ii) boiled enzyme as a control assay, (iii) TE1, (iv) TE1-TE2* that carries 3



Page 4 of 15

a Ser-to-Ala mutation in TE2, (v)TE1*-TE2 that carries a Ser-to-Ala mutation in TE1, and (vi) TE1-TE2 that carries a double Ser-to-Ala mutation in both TE1 and TE2. Red asterisks represent mutation of the key Ser residues. All the enzymes produced the hydrolyzed product that is 3～ 5-fold higher than P1. We did not get soluble TE2 for an enzymatic assay. A full set of chromatograms showing the relative intensity of the substrate 1, the cyclized product P1, and the hydrolyzed product is shown in Figure S4.

Tandem TEs have been found in several NRPS systems.28-32 To reveal the phylogeny of the two TEs in teixobactin biosynthesis, we constructed a Bayesian phylogenetic tree using different TEs reported in the literature (Figure S5 and Table S3). This analysis showed that the two teixobactin TEs share the closest relationship with the two TE domains in lysobactin biosynthesis (Figure S3).28 Lysobactin and teixobactin are very similar in both structure and mode of action: both compounds are depsipeptides consisting of 11 amino acid (aa) residues (Figure S6) and both bind lipid II.1,33 The function of the tandem TE in lysobactin biosynthesis (TE1lyb and TE2lyb) has been investigated by Marahiel and coworkers, showing that TE1lyb is solely responsible for lactonization that releases lysobactin, whereas TE2lyb mediates the hydrolysis of the misprimed carrier protein in the NRPS assembly line.28 Because the teixobactin TEs are phylogenetically closely-related to their lysobactin counterparts, we initially believed that the two tandem TE systems share a similar mechanism. To test this hypothesis, we expressed TE1 separately with an N-terminal hexa-histidine tag and incubated it with 1. However, although TE1 produced the hydrolyzed product similar to that of TE1-TE2, production of the cyclized product P1 was not observed in the reaction (Figure 2, trace iii), suggesting that, unlike TE1lyb, the teixobactin TE1 alone is not able to catalyze the lactonization reaction.

Marahiel and coworkers showed that TE1lyb-TE2lybwas partially proteolytically degraded during expression, resulting in the simultaneous purification of the full TE1lyb-TE2lyb as well as TE1lyb.28 Because most of the known TEs that serve proofreading functions are standalone proteins, proteolysis of TE1lyb-TE2lyb that released TE2lyb as a separate protein during expression suggests that TE2lyb might be a proofreading TE. This observation is consistent with the proposed 4


Page 5 of 15 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


proofreading function of TE2lyb, which is unrelated to the reaction catalyzed by TE1lyb. However, proteolytic degradation of the teixobactin TE1-TE2 was not observed in the expression and purification process. We were also unable to express TE2 separately in soluble form despite different efforts. To dissect the mechanism of the tandem TE-catalyzed lactonization reaction, we mutated the Ser residue in the conserved GXSXG (GHSFG) motif of TE2 (Figure 3) to Ala and expressed the resulting TE1-TE2* (* denotes a Ser-to-Ala mutation in the GXSXG motif of a TE domain;34-36 the same denotation is also used below). LC-HRMS analysis of the reaction mixture showed that TE1-TE2* converted 1 to the expected product P1 with an efficiency similar to the wild type TE1-TE2 (Figure 2, trace iv, and Table S1). Unexpectedly, when we replaced the Ser residue in the conserved GXSXG (GWSGG) motif of TE1 (Figure 3) with Ala and generated TE1*-TE2, we found that this protein also produced P1 from 1 with a similar efficiency (Figure 2, trace v). However, when we mutated the two active site Ser residues, the lactonization activity was completely abolished in the resulting TE1*-TE2* protein (Figure 2, trace vi).

Figure 3. Sequence alignment of TE1 and TE2 with other selected TEs whose crystal structures have been solved. These TEs are involved in fengycin biosynthesis (FenTE)30, erythromycin biosynthesis (EryTE)29, and enterobactin biosynthesis (EntTE)31, respectively. The key Ser residue is shown by a red asterisk, and the conserved GxSxG motif in TE domains is shown by a blue bar.

The results presented above suggest that the two teixobactin domains function together in catalyzing the lactonization step. Unlike lysobactin biosynthesis, where TE1lyb and TE2lyb are responsible for cyclization and proofreading, respectively,28 the two TEs in teixobactin biosynthesis are functionally exchangeable in catalyzing the lactonization reaction (Figure 2). Because TE1 is not able to produce the cyclized product whereas TE2 was not expressed in soluble form, both TEs are likely essential for the activity, which possibly form together an active 5



Page 6 of 15

site cavity for linear peptide cyclization. A possible mechanistic scheme for the synergistic action of TE1 and TE2 is shown in Figure 4. We would like to point out that it is also possible that each TE can be active without needing any synergy, but the isolated TE domain cannot be folded properly in the absence of the second counterpart.

Se r2

Se r2

Figure 4. A mechanistic proposal for the synergistic action of TE1 and TE2 in the lactonization reaction. TE1 and TE2 together form an active site cavity that is essential for cyclization. The two domains are functionally exchangeable and both Ser residues in TE1 and TE2 can be acylated for the cyclization the reaction.

We next investigated the substrate specificity of the TE-catalyzed cyclization reaction. To investigate the stereochemistry in enzyme recognition, we synthesized a linear peptide 2 that contains only one D-aa (D-Thr8) whereas all other D-aa residues in 1 were replaced by their L-type isomers (Table 1, entry 2, and Figure S7). Biochemical analysis showed that TE1-TE2 produced the cyclized product P2 from 2 with an efficiency similar to P1 production (Table S1), suggesting that the stereochemistry of other D-aa residues is not important for enzyme activity. We also replaced the L-Lys10 of 2 with L-Arg and found the resulting substrate 3 was readily converted to the expected cyclized product P3 (Table 1, entry 3, and Figure S8). However, when we changed the D-Thr8 of 3 to L-Thr and produced 4, production of the expected cyclized product was not observed in this reaction (Table 1, entry 4), suggesting that the D-configuration of the to-be-cyclized Thr8 is strictly essential for enzyme activity. We also replaced the D-Thr8 of 3 with D-Ser, and found that the resulting peptide 5 was converted to the expected cyclized product P5 (Table 1, entry 5, and Figure S9). A truncated peptide 6 lacking the N-terminal three residues was 6


Page 7 of 15 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


also synthesized, which was also readily converted to the expected cyclized product P6 (Table 1, entry 6, and Figure S10), suggesting that the N-terminal part of peptide substrates is not necessary for enzyme recognition.

In summary, we have established a TE-based chemoenzymatic approach for the synthesis of teixobactin analogues. The tandem TE has a relaxed substrate specificity, allowing for the production of a series of teixobactin analogues, which complements the recent chemical synthesis efforts. More intriguingly, we found that, unlike lysobactin biosynthesis in which only one TE (i.e. TE1lys) is responsible for lactonization reaction,28 the two teixobactin TE domains works together and are functionally exchangeable. The close phylogeny but very distinct TE mechanisms observed in lysobactin and teixobactin biosynthesis demonstrates the remarkable mechanistic diversity in NRPS enzymology and the potential to engineer these megasynthases for novel activities. Using of the synthetically more accessible methyl esters in this study also establishes a more convenient way to investigate the in vitro activity of TEs.

7



Page 8 of 15

Table 1. Substrate specificity of the TE-mediated lactonization reaction. The cyclization sites are shown in blue, and other D-aa residues were shown in cyan. The chemical structures of the cyclized products are shown in Figure S11. For the detailed MS/MS spectra of each cyclized product, please see Figure S2 for P1 and Figure S5-S8 for P2, P3, P5 and P6 in the Supporting Information. Linear Substrate

Cyclized Product

Not observed

Experimental Section Instrumentation and materials High-performance liquid chromatography (HPLC) was performed using a Thermo Scientific Dionex Ultimate 3000 system with a diode array detector. High resolution mass spectra (HRMS) were acquired using a Q-Exactive™ Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher) equipped with a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific Inc.). PCR was performed on a Bio-Rad T100TM Thermal Cycler using Phanta® Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co. Ltd, China). All chemical reagents and anhydrous solvents were purchased from commercial sources and used without further purification unless otherwise specified.

Peptide Synthesis The linear C-terminal methyl ester peptides (1-6) were synthesized through standard SPPS method by using the synthetic procedure detailed in in the Supporting Information. 8


Page 9 of 15 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


Plasmid construction Codon-optimized DNA encoding TE1-TE2 was synthesized from GENEWIZ (China) and was cloned into the EcoRI and HindIII restriction sites of pET28a (Novagen), generating the expression plasmid pTE1-TE2-ET28a. Using this plasmid as a template, two fragments were amplified by a primer pair TE1-(EcoRI)-FOR and TE1-(HindIII)-REV, and a primer pair TE2-(EcoRI)-FOR and TE2-(HindIII)-REV, respectively (see Table S1 for the primer sequences). The resulting DNA fragments were digested by EcoRI and HindIII, and were inserted into the same site of pET28a to generate pTE1-ET28a (for expressing TE1) and pET2-ET28a, respectively.

To construct the plasmid for expressing TE1*-TE2, two fragments were amplified by a primer pair TE1-(EcoRI)-FOR and TE1-S97A-REV, and a primer pair TE1-S97A-FOR and TE2-(HindIII)-REV, respectively (see Table S1 for the primer sequences), using pTE1-TE2-ET28a as the template. The resulting PCR products were cloned into pET28a digested with EcoRI/HindIII by homologous recombination, using ClonExpressMultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd). Briefly, 15 µL mixture of 3 µL 5×CE MultiS Buffer, 1 µL Exnase MultiS, 100 ng linear plasmid, 15 ng fragment 1 and fragment 2 and ddH2O was incubated at 37℃ for 30 min. Chemically competent E. coli DH5α cells were transformed with the ligation mixture and plated on LB-agar containing kanamycin (50 µg/mL) to screen for positive clones. The resulting plasmid pTE1*-TE2-ET28a was confirmed by DNA sequencing.

To construct the plasmid for expressing TE1-TE2*, two fragments were amplified by a primer pair TE1-(EcoRI)-FOR

and

TE2-S377A-REV,

and

a

primer

pair

TE2-S377A-FOR

and

TE2-(HindIII)-REV, respectively (see Table S1 for the primer sequences), using pTE1-TE2-ET28a as the template. The resulting PCR products were cloned into pET28a digested with EcoRI/HindIII by homologous recombination as described above. The resulting plasmid pTE1-TE2*-ET28a was confirmed by DNA sequencing.

To construct the plasmid for expressing TE1-TE2*, two fragments were amplified by a primer pair 9



TE1-(EcoRI)-FOR TE2-(HindIII)-REV,

and

TE2-S377A-REV,

respectively

(see

and

Table

a S1

primer for

the

Page 10 of 15

pair

TE2-S377A-FOR

primer

sequences),

and using

pTE1*-TE2-ET28a as the template. The resulting PCR products were cloned into pET28a digested with EcoRI/HindIII by homologous recombination as described above. The resulting plasmid pTE1-TE2*-ET28a was confirmed by DNA sequencing.

Protein expression and purification E. coli BL21 (DE3) cells were transformed via electroporation with each expression plasmid. A single colony transformant was used to inoculate a 5 mL culture of LB supplemented with 50 µg/mL kanamycin. The culture was grown at 37 °C for 12 h and was used to inoculate 1 L of LB-medium containing 50 µg/mL kanamycin. Cells were grown at 37°C and 180 rpm to an OD600～0.6-0.8, and then IPTG was added to a final concentration of 0.2 mM. After additional 18 h of incubation at 20 °C and 180 rpm, the cells were harvested by centrifugation at 5000 rpm for 15 min at 4 °C. The pellet was used directly for targeted protein purification or stored at −80 °C upon further use.

The cell pellet was resuspended in 20mL of the lysis buffer (50 mM MOPS, 200 mM NaCl, and 10% glycerol, pH 8.0), and was lysed by sonication on ice. Cell debris was removed via centrifugation at 14000 rpm for 30 min at 4 °C. The supernatant was incubated with 4 ml Ni-NTA resin pre-equilibrated with the lysis buffer, and then subjected to affinity purification on a column. The desired fractions were combined and concentrated using an Amicon Ultra-15 Centrifugal Filter Unit and the concentrated protein solution was desalted using a PD-10 column (GE Healthcare) pre-equilibrated with the elution buffer I (50 mM MOPS, 25 mM NaCl, 10 mM DTT and 10% (v/v) glycerol, pH 8.0). The protein fraction was collected and concentrated, analyzed by SDS-PAGE (12% Tris-glycine gel), and was used directly for in vitro assay or stored at −80 °C upon further use. Protein concentration was determined using a Bradford Assay Kit (Bio-Rad) using bovine serum albumin (BSA) as a standard.

Enzymatic assays A typical assay was carried out by incubating 200 µM precursor peptides (1-6) with ～10 µM 10


Page 11 of 15 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


protein (TE1-TE2, TE1, TE1*-TE2, TE1-TE2*, or TE1*-TE2*) in 20 mM Tris buffer (pH 8.0). Reaction volumes were typically 100 µL and were maintained at room temperature for 1 h prior to quenching. The reactions were quenched by addition of two-volume of methanol. After removal of the protein precipitates by centrifugation, the supernatant was subjected to LC-MS analysis. For kinetic analysis, reactions were carried out using 200 µM or 400 µM 1 or 2, and the reactions were quenched at 10 min, 20 min, 30 min, and 60 min, respectively. Production was roughly estimated according to MS intensity.

LC-HRMS analysis LC-HRMS and MS/MS analysis was carried out on a Q-Exactive™ Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher) equipped with a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific Inc.) The column(BioBasic-8, Thermo Scientific, 2.1 x 100 mm,5 um particle size) was equilibrated with 98% solvent A (ddH2O + 0.1% formic acid) and 2% solvent B (acetonitrile +0.1% formic acid), and developed at a flow rate of 0.3 mL/min: 0-2 min, constant 98%A / 2% B; 2-8.5 min, linear gradient from 98% A / 2% B to 5% A / 95% B; 8.5-11.5 min, constant 5%A / 95% B; 11.5-12.5, a linear gradient to 98% A / 2% B. Detection range was set to a mass to charge ratio (m/z) from 133 to 2000. P1, HRMS (ESI-TOF) m/z: [M + 2H]++ Calcd for C57H97N13O15 (2z) 601.8613; Found 601.8607; P2, HRMS (ESI-TOF) m/z: [M + 2H]++ Calcd for C57H97N13O15 (2z) 601.8613; Found 601.8602; P3, HRMS (ESI-TOF) m/z: [M + 2H]++ Calcd for C57H97N15O15 (2z) 615.8644; Found 615.8634; P5, HRMS (ESI-TOF) m/z: [M + 2H]++ Calcd for C56H95N15O15 (2z) 608.8566; Found 608.8565; P6, HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C39H71N12O11 883.5365; Found 883.5348.

Phylogenetic analysis Bayesian MCMC inference of TE phylogeny was performed using the program MrBayes (version 3.2)37 with sequences listed in Table S2. Final analyses consisted of two sets of four chains each (one cold and three heated), run for about 1 million generations with trees saved and parameters sampled every 100 generations. Analyses were run to reach a convergence with SD of split frequencies < 0.01. Posterior probabilities were averaged over the final 75% of trees (25% burn in). 11



Page 12 of 15

ASSOCIATED CONTENT: Supporting Information: Descriptions of methods for site-directed mutagenesis, phylogenetic study, and LC-HRMS analysis, and supporting figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION: Corresponding Author: Qi Zhang *E-mail: [email protected]. ORCID Qi Zhang: 0000-0002-8135-2221

Notes The authors declare no competing financial interest

Acknowledgments: This work was supported by grants from the National Key R&D Program of China (2016 Y F A0501302), from the National Natural Science Foundation of China (31500028 and 31670060 to Q.Z., and 31600398 to W.D.), and from the Open Fund of Key Laboratory of Glycoconjugate Research, Fudan University, Ministry of Public Health.

References (1) Ling, L. L.; Schneider, T.; Peoples, A. J.; Spoering, A. L.; Engels, I.; Conlon, B. P.; Mueller, A.; Schaberle, T. F.; Hughes, D. E.; Epstein, S.; Jones, M.; Lazarides, L.; Steadman, V. A.; Cohen, D. R.; Felix, C. R.; Fetterman, K. A.; Millett, W. P.; Nitti, A. G.; Zullo, A. M.; Chen, C.; Lewis, K. Nature 2015, 517, 455. (2) Homma, T.; Nuxoll, A.; Gandt, A. B.; Ebner, P.; Engels, I.; Schneider, T.; Gotz, F.; Lewis, K.; Conlon, B. P. Antimicrob Agents Chemother 2016, 60, 6510. (3) Liu, Y.; Chan-Park, M. B.; Mu, Y. Sci Rep 2017, 7, 17197. (4) Guo, C.; Mandalapu, D.; Ji, X.; Gao, J.; Zhang, Q. Chemistry 2017, doi: 10.1002/chem.201704167. 12


Page 13 of 15 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


(5) von Nussbaum, F.; Sussmuth, R. D. Angew Chem Int Ed Engl 2015, 54, 6684. (6) Piddock, L. J. J Antimicrob Chemother 2015, 70, 2679. (7) Wright, G. Nature 2015, 517, 442. (8) Fiers, W. D.; Craighead, M.; Singh, I. ACS Infect Dis 2017, 3, 688. (9) Jad, Y. E.; Acosta, G. A.; Naicker, T.; Ramtahal, M.; El-Faham, A.; Govender, T.; Kruger, H. G.; de la Torre, B. G.; Albericio, F. Org Lett 2015, 17, 6182. (10) Giltrap, A. M.; Dowman, L. J.; Nagalingam, G.; Ochoa, J. L.; Linington, R. G.; Britton, W. J.; Payne, R. J. Org Lett 2016, 18, 2788. (11) Jin, K.; Sam, I. H.; Po, K. H.; Lin, D.; Ghazvini Zadeh, E. H.; Chen, S.; Yuan, Y.; Li, X. Nat Commun 2016, 7, 12394. (12) Parmar, A.; Iyer, A.; Vincent, C. S.; Van Lysebetten, D.; Prior, S. H.; Madder, A.; Taylor, E. J.; Singh, I. Chem Commun (Camb) 2016, 52, 6060. (13) Yang, H.; Chen, K. H.; Nowick, J. S. ACS Chem Biol 2016, 11, 1823. (14) Monaim, S. A. H. A.; Jad, Y. E.; Acosta, G. A.; Naicker, T.; Ramchuran, E. J.; El-Faham, A.; Govender, T.; Kruger, H. G.; de la Torre, B. G.; Albericio, F. Rsc Advances 2016, 6, 73827. (15) Abdel Monaim, S. A. H.; Jad, Y. E.; Ramchuran, E. J.; El-Faham, A.; Govender, T.; Kruger, H. G.; de la Torre, B. G.; Albericio, F. ACS Omega 2016, 1, 1262. (16) Parmar, A.; Prior, S. H.; Iyer, A.; Vincent, C. S.; Van Lysebetten, D.; Breukink, E.; Madder, A.; Taylor, E. J.; Singh, I. Chem Commun (Camb) 2017, 53, 2016. (17) Parmar, A.; Iyer, A.; Lloyd, D. G.; Vincent, C. S.; Prior, S. H.; Madder, A.; Taylor, E. J.; Singh, I. Chem Commun (Camb) 2017, 53, 7788. (18) Yang, H.; Du Bois, D. R.; Ziller, J. W.; Nowick, J. S. Chem Commun (Camb) 2017, 53, 2772. (19) Jin, K.; Po, K. H. L.; Wang, S.; Reuven, J. A.; Wai, C. N.; Lau, H. T.; Chan, T. H.; Chen, S.; Li, X. Bioorg Med Chem 2017, 25, 4990. (20) Abdel Monaim, S. A. H.; Ramchuran, E. J.; El-Faham, A.; Albericio, F.; de la Torre, B. G. J Med Chem 2017, 60, 7476. (21) Schumacher, C. E.; Harris, P. W. R.; Ding, X. B.; Krause, B.; Wright, T. H.; Cook, G. M.; Furkert, D. P.; Brimble, M. A. Org Biomol Chem 2017, 15, 8755. (22) Abdel Monaim, S. A. H.; Jad, Y. E.; El-Faham, A.; de la Torre, B. G.; Albericio, F. Bioorg Med Chem 2017, doi: 10.1016/j.bmc.2017.09.040. (23) Keating, T. A.; Ehmann, D. E.; Kohli, R. M.; Marshall, C. G.; Trauger, J. W.; Walsh, C. T. Chembiochem 2001, 2, 99. (24) Yeh, E.; Lin, H.; Clugston, S. L.; Kohli, R. M.; Walsh, C. T. Chem Biol 2004, 11, 1573. (25) Lin, H.; Walsh, C. T. J Am Chem Soc 2004, 126, 13998. (26) Koketsu, K.; Oguri, H.; Watanabe, K.; Oikawa, H. Chem Biol 2008, 15, 818. (27) Robbel, L.; Hoyer, K. M.; Marahiel, M. A. FEBS J 2009, 276, 1641. (28) Hou, J.; Robbel, L.; Marahiel, M. A. Chem Biol 2011, 18, 655. (29) Scholz-Schroeder, B. K.; Soule, J. D.; Gross, D. C. Mol Plant Microbe Interact 2003, 16, 271. (30) Roongsawang, N.; Washio, K.; Morikawa, M. Chembiochem 2007, 8, 501. (31) de Bruijn, I.; de Kock, M. J.; de Waard, P.; van Beek, T. A.; Raaijmakers, J. M. J Bacteriol 2008, 190, 2777. (32) Biggins, J. B.; Kang, H. S.; Ternei, M. A.; DeShazer, D.; Brady, S. F. J Am Chem Soc 2014, 13



Page 14 of 15

136, 9484. (33) Lee, W.; Schaefer, K.; Qiao, Y.; Srisuknimit, V.; Steinmetz, H.; Muller, R.; Kahne, D.; Walker, S. J Am Chem Soc 2016, 138, 100. (34) Tsai, S. C.; Miercke, L. J.; Krucinski, J.; Gokhale, R.; Chen, J. C.; Foster, P. G.; Cane, D. E.; Khosla, C.; Stroud, R. M. Proc Natl Acad Sci U S A 2001, 98, 14808. (35) Samel, S. A.; Wagner, B.; Marahiel, M. A.; Essen, L. O. J Mol Biol 2006, 359, 876. (36) Frueh, D. P.; Arthanari, H.; Koglin, A.; Vosburg, D. A.; Bennett, A. E.; Walsh, C. T.; Wagner, G. Nature 2008, 454, 903. (37) Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D. L.; Darling, A.; Hohna, S.; Larget, B.; Liu, L.; Suchard, M. A.; Huelsenbeck, J. P. Syst Biol 2012, 61, 539.

14


Page 15 of 15 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


Table of Contents (TOC) graphic

O OMe

TE1-TE2 or TE1*-TE2 or TE1-TE2* O

OH

TE1*-TE2*

O

teixobactin analogues

(* represents a Ser-to-Ala mutation of the key Ser in a TE domain)

15


Thioesterase-Mediated Synthesis of Teixobactin Analogues

Recommend Documents