Novel chemical probes for the investigation of nonribosomal peptide assembly

Article abstract of DOI:10.1039/c7cc02427d

Chemical probes were devised and evaluated for the capture of biosynthetic intermediates involved in the bio-assembly of the nonribosomal peptide echinomycin. Putative intermediate peptide species were isolated and characterised, providing fresh insights

Full text of DOI:10.1039/c7cc02427d

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Novel chemical probes for the investigation
of nonribosomal peptide assembly†
Cite this:DOI: 10.1039/c7cc02427d
Y. T. Candace Ho,a Daniel J. Leng,a Francesca Ghiringhelli,ab Ina Wilkening,a
Dexter P. Bushell,a Otto Kostner,ac Elena Riva,a Judith Havemann,a
Received 30th March 2017,
Accepted 12th June 2017
¨
b
a
Daniele Passarella
and Manuela Tosin
*
DOI: 10.1039/c7cc02427d
rsc.li/chemcomm
Chemical probes were devised and evaluated for the capture of
biosynthetic intermediates involved in the bio-assembly of the
nonribosomal peptide echinomycin. Putative intermediate peptide
species were isolated and characterised, providing fresh insights
into pathway substrate flexibility and paving the way for novel
chemoenzymatic approaches towards unnatural peptides.
Peptidic molecules are the most abundant and versatile chemical
entities in nature: from proteins to small peptides, they display
countless architectures and exert key roles in almost every known
biological process. In the context of secondary metabolism,
peptide natural products comprise a vast array of bioactive
molecules that regulate interspecies communication and organism
survival. Peptide natural products can be either biosynthesised by
the ribosome (and hence known as ribosomal peptides, RiPPs)
or by the multifunctional enzymes nonribosomal peptide
synthetases (NRPSs).1
Fig. 1 (A) General mechanism of nonribosomal peptide assembly;
(B) proposed capture of peptide biosynthetic intermediates (2) via newly
devised chain termination probes (1) based on nonhydrolysable analogues
(X = NH) of PCP-bound amino acids. PCP = peptidyl carrier protein;
C = condensation domain; d = aminoacyl donor site; a = aminoacyl acceptor
site. R3 = variable alkyl moiety; R4 = variable amino acid side chain.
Amongst NRPs we encounter potent anticancer agents, such
as bleomycin, and antibiotics of last resort, such as vancomycin in product structure and bioactivity. Key domains utilised in
and teicoplanin.2 In NRP biosynthesis aminoacyl units, anchored peptide elaboration throughout assembly include methyl-
as thioesters to peptidyl carrier proteins (PCPs) via the phospho- transferases, epimerases4 and heterocyclases, whereas tailoring
pantetheine cofactor,3 are joined together through peptide bond enzymes acting in post-NRPS processes comprise oxidases,5
formation by condensation (C) domains (Fig. 1A). Similarly to halogenases6 and glycosyltransferases.7
polyketide and fatty acid synthases (PKSs and FASs, respectively),
In NRPS assembly, adenylation (A) domains act as ‘gate-keepers’
NRPSs generate growing enzyme-bound biosynthetic intermedi- by selecting specific amino acids and activating them as adenosine
ates which are variably processed and ultimately converted to the monophosphate (AMP) esters for their loading onto the phospho-
final products. The ability of NRPSs to process proteinogenic and pantetheine cofactors of PCPs.8 C domains catalyse peptide bond
non-proteinogenic amino acids, fatty acids and a-hydroxy acids, formation and present two distinct substrate-binding sites: a ‘donor
linking them in different ways, give rise to astonishing diversity site’ for upstream PCP-bound amino acids, and an ‘acceptor site’ for
downstream PCP-bound amino acids; the free amino groups of the
latter act as nucleophiles towards the thioester moiety of PCP-bound
upstream substrates to generate extended PCP-bound intermediates
(Fig. 1A), which are subsequently transferred to other sites for
further extension and elaboration.9 Eventually peptide chain assem-
bly is terminated, mostly by thioesterase (TE) domains promoting
peptide hydrolysis or cyclisation;10 additional mechanisms of
peptide chain termination and release include reduction of the
final thioester bond to an aldehyde or an alcohol by R domains.11
a Department of Chemistry, University of Warwick, Library Road, CV4 7AL, UK.
E-mail: M.Tosin@warwick.ac.uk; Tel: +442476572878
b Department of Chemistry, Universita’ degli Studi di Milano, Via Golgi,
19 20133 Milano, Italy
c
¨
¨
¨
Institut fur Organische Chemie, Universitat Wien, Wahringer Str., 38 1090 Wien,
Austria
† Electronic supplementary information (ESI) available: General methods for the
synthesis of chemical probes and LC-HRMSn analysis of the biosynthetic inter-
mediates isolated from S. lasaliensis strains. See DOI: 10.1039/c7cc02427d
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Table 1 Chemical probes prepared for the capture of peptide biosyn-
thetic intermediates in echinomycin bio-assembly (see Fig. 2)
As PKSs and FASs, NRPSs can be modular or iterative enzymes: the
former comprise multiple sets of domains (modules), with each
module catalysing at least one round of peptide chain extension and
a direct correspondence between protein predicted function and
product in most cases.12 Conversely, iterative synthases are con-
stituted by modules that are used more than once to catalyse
peptide chain growth and processing,13 hence the nature of their
products is not fully predictable.
The engineering of NRPS enzymes and pathways remains a major
synthetic biology focus in view of novel peptide production.14–17
Despite the much-improved ability to engineer NRPSs, many details
on peptide assembly catalysed by these enzymes remain unknown,
limiting our ability to fully exploit existing NRPSs and to design
de novo enzymes of improved versatility and efficiency.
Compound
number
R3
Probe structure
Captured species
CH3
3
4
5
n.d.
(CH2)2CH3
(CH2)5CH3
Dipeptideb
Di-, tri-, tetrapeptideb
n.d.
(CH2)8CH3 6c
(CH2)2CH3
(CH2)5CH3
7
8
Di-, tripeptideb
Di-, tri-, tetrapeptideb
(CH2)5CH3
9
Di-,a tripeptideb
Dipeptidea, tri-, tetra-,
pentapeptidesb
Dipeptidea, tri-,
tetrapeptidesb
n.d.
Over the years mechanistic insights on NRP assembly have been
gathered from isotopic labelling of aminoacyl building blocks,18
genetic manipulation of in vivo biosynthetic pathways (e.g. by
domain inactivation, deletion, etc.),19 NRPS activity reconstitution
in vitro,11a,20 mass spectrometry of metabolites and enzyme-bound
precursors,20c,21 and enzyme conformational/structural elucidation.22
Nonetheless we still lack a comprehensive and dynamic picture of
how NRPS machineries work in vivo as a whole in processing
substrates and successfully conveying intermediates to the end of
a biosynthetic process. A major hurdle in gathering this information
is given by the covalent tethering of NRP intermediates to their
biosynthetic enzymes throughout peptide assembly.
In the past few years our group has developed ‘chain termination’
probes for the investigation of polyketide biosynthesis: nonhydrolys-
able small molecule mimics of malonate building blocks recruited in
polyketide formation were devised to interfere in the key decarbox-
ylative Claisen condensation step leading to polyketide chain assem-
(CH2)2CH3 10
(CH2)5CH3 11
(CH2)8CH3 12c
Dipeptidea, tri-,
tetrapeptideb
Dipeptidea,
tripeptideb
n.d.
(CH2)2CH3 13
(CH2)5CH3 14
(CH2)8CH3 15
(CH2)5CH3 16
(CH2)5CH3 17
Dipeptideb
n.d.
a
b
c
Major species. Detected in minor amounts (see ESI). Displaying
bly, thereby ‘capturing’ transient biosynthetic intermediates for cytotoxicity above 1 mM.
characterisation.23–26 These tools have proved successful for inter-
mediate isolation and characterisation of intermediate species from that, once within NRPS active sites, these molecules should be
modular23b,24,26 and iterative PKS enzymes,23a,25 gathering in vitro and recognised as acceptor substrates by C domains and hence compete
in vivo key information on the timing and the mechanism of catalytic with PCP-bound aminoacyl units for peptide bond formation,
events otherwise inaccessible, and unveiling novel opportunities for ultimately resulting in the off-loading and capture of prema-
natural product diversification.24c
turely truncated peptides (2, Fig. 1B).
Given the similarity between PKSs and NRPSs in processing
We initially prepared N-acetyl substrates based on cognate amino-
carrier protein-bound species, we reasoned that chemical probes acyl moieties such as the alanine derivative 3 (Table 1) via standard
mimicking PCP-bound amino acids could be developed to interfere peptide synthesis procedures (ESI†). These molecules were adminis-
in NRPS-catalysed peptide bond formation and capture intermediate tered in variable concentrations to liquid and solid cultures of
peptide species for further characterisation. Hence we prepared a S. lasaliensis ACP12 (S970A), an engineered strain of S. lasaliensis
library of putative probes (3–17, Table 1) for the investigation of NRRL 3382R incapable of producing the polyketide lasalocid A24a but
echinomycin assembly in the soil bacterium S. lasaliensis. Echinomycin still capable of generating the nonribosomal peptide echinomycin in
(18, Fig. 2) is an antitumor antibiotic that acts as DNA bis-intercalator relevant levels. Preliminary LC-HRMSn analysis of organic extracts of
due to its two-fold symmetry and quinoxaline-derived moieties. It is microbial fermentations showed that the probes were present and
biosynthesised by an iterative NRPS that assembles two identical hydrolytically stable but no evidence of feasible intermediates (data
peptide chains from 2-quinoxaline carboxylic acid, L-serine, alanine, not shown). We reasoned that perhaps the hydrophilicity of peptide
cysteine and valine (Fig. 2): a terminal thioesterase domain catalyses intermediates could hamper their isolation via organic extraction.
depsipeptide formation and lactonisation, followed post-NRPS oxida-
tive processing.27
Therefore we prepared probes of variable N-acyl chain length
(R3, Fig. 1 and Table 1) in order to increase their hydrophobicity and
The probes devised by us were N-acyl cysteamine derivatives28 that cellular uptake. Besides, variable side chain moieties (variable R4,
mimic PCP-bound aminoacyl moieties and feature nonhydrolysable Fig. 1) and amino acid scaffold motifs (e.g. a versus b-amino acids,
amide bonds in place of cleavable thioester bonds (1, X = NH, Fig. 1) Table 1) were also included in the probe structure in order to assess
in order to prevent substrate re-loading onto NRPSs. We reasoned the substrate flexibility of the echinomycin NRPS machinery in vivo.
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Fig. 2 (A) Overview of echinomycin nonribosomal peptide assembly and in vivo capture of putative biosynthetic intermediates via chemical probes 3–17
(note: the general intermediate structures apply to all substrates with the exception of b-alanine-based probes 13–15, ESI†); (B) extracted ion
chromatogram of a dipeptide resulting from the capture of quinoxaline 2-carboxylic acid by probe 11 (observed MS2 fragments indicated, ESI†); (C) HR-
MS2 analyses of a tetrapeptide intermediate resulting from the capture of an enzyme-bound tripeptide by probe 8. The stereochemistry of captured
intermediates is yet to be established.
An overview of echinomycin putative intermediates captured from amino acids. Variants of nonribosomal peptides resulting from the
S. lasaliensis ACP12 (S970A) via the newly devised chemical probes is incorporation of different amino acids can be observed in vivo,9b and
given in Table 1 and Fig. 2. A whole range of captured species this has been utilised for precursor-directed biosynthesis purposes.14
spanning from dipeptides to pentapeptides (whose putative struc- However, to the best of our knowledge, the current study constitutes
tures are represented in Fig. 2) were isolated and characterised by the first report of in vivo probing of nonribosomal peptide assembly
HR-MSn: these were identified as putative echinomycin intermediates utilising aminoacyl N-acetylcysteamine substrate mimics.
by MSn fragment peaks (obtained from amide cleavages) featuring
The overall results gathered by us seem to indicate that: (1) the
quinoxaline 2-carboxylic acid and other amino acid constituents of echinomycin biosynthetic machinery possesses some flexibility
echinomycin in the expected sequence/order (as shown in Fig. 2C towards the processing of ‘unnatural’ substrates in the correspon-
and in the ESI†). The putative species were absent in control samples dence of specific C domains, possibly due to flexible pseudo-
and substantially varied in amount and distribution according to the substrate positioning at the enzyme active site during peptide bond
probe utilised (ESI†).
formation9d and/or probe bioavailability in vivo: this seems particu-
Besides the expected species captured from probes based on larly true for Gly and b-alanine substrates, which lack side-chain
cognate substrates, additional species deriving from non-cognate stereochemistry and steric hindrance; (2) dipeptides accumulate
pseudo-substrates were detected (Table 1, Fig. 2 and the ESI†). For preferentially in comparison to more advanced intermediate species
instance, dipeptides allegedly deriving from the off-loading of the (see ESI† figures): this suggests that the first condensation step
starter quinoxaline 2-carboxylic acid were detected in significant might be the slowest amongst all those taking place throughout
amounts from experiments utilising non-cognate glycine (Fig. 2B) echinomycin peptide chain assembly.
and b-alanine probes (Fig. 30S, ESI†) as well as the cognate serine
A more in-depth assessment and dissection of these in vivo
substrates (Fig. 17S, ESI†). Further advanced species (from tri- to findings will require separate in vitro experiments with recombi-
pentapeptides) were most efficiently detected and characterised in nant C domains, as well as the development of advanced
extracts deriving from bacterial fermentations in the presence of analytical tools29 capable of deconvoluting the acquired LC-MS
N-butyroyl and N-heptanoyl glycine (Fig. 21S–23S, 26S and 27S, ESI†), data in a quantitative fashion. Nonetheless the preliminary
alanine (Fig. 8S, ESI†), b-alanine (Fig. 31S and 32S, ESI†), and valine experiments herein reported demonstrate that the in vivo
probes (Fig. 2C). No intermediate species were captured utilising the profiling of NRP assembly via chain termination probes is now
aromatic L-phenylalanine pseudo-substrate 17.
possible, with important implications for future biosynthetic
Aminoacyl N-acetylcysteamine (SNAc) thioesters28 have been often pathway engineering. The screening of natural product bio-
utilised to reconstitute the activity of C domains in vitro and have assembly can indeed provide not only preliminary information
shown that C acceptor sites generally exhibit strong stereoselectivity on substrate recognition but also insights on the kinetics of
(L- versus D-), together with some selectivity towards the side chain of natural product assembly,26 constituting the rational for
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peptide production.
In summary, we have herein gathered a first direct view of
substrate processing for an iterative NRPS in vivo through the use
of newly devised nonhydrolysable ‘chain termination’ probes.
Further applications of these tools for the investigation of nonribo-
somal peptide pathways will be reported in due course.
We gratefully acknowledge BBSRC (project grant BB/J007250/1 to
M. T. and MIBTP studentship to D. J. L.); the Erasmus programme
(exchange bursaries to F. G. and O. K.); FP7 (Marie Curie Intraeur-
opean Fellowship to I. W.); the Institute of Advanced Studies at
Warwick (Postdoctoral Fellowship to E. R.); Dr Cleidiane Zampronio
(School of Life Sciences, Warwick) for assistance with LC-HRMSn
Orbitrap Fusion analyses; Dr Lijiang Song for preliminary MS data
acquired on a Bruker MaXis Impact instrument; Prof. Peter F. Leadlay
(Cambridge) for the kind gift of S. lasaliensis ACP12 (S970A); and
COST Action CM1407 for networking funding and opportunities.
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