Available online at www.sciencedirect.
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Nonribosomal peptide synthetases: structures and dynamics
Matthias Strieker, Alan Tanović and Mohamed A Marahiel
Nonribosomal peptide synthetases (NRPSs) are large
multimodular biocatalysts that utilize complex regiospecific
and stereospecific reactions to assemble structurally and
functionally diverse peptides that have important medicinal
applications. During this ribosome-independent peptide
synthesis, catalytic domains of NRPS select, activate or modify
the covalently tethered reaction intermediates to control the
iterative chain elongation process and product release. Recent
advances in structural elucidation of domains, didomains, and
an entire termination module revealed valuable insights into the
mechanism of nonribosomal synthesis and are highlighted
herein.
Address
Philipps-University Marburg, Department of Chemistry/Biochemistry,
Hans-Meerwein-Strasse, D-35032 Marburg, Germany
Corresponding author: Marahiel, Mohamed A
(marahiel@staff.uni-marburg.de)
Current Opinion in Structural Biology 2010, 20:234–240
This review comes from a themed issue on
Macromolecular assemblages
Edited by Edward H Egelman and Nenad Ban
Available online 10th February 2010
0959-440X/$ – see front matter
# 2010 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.sbi.2010.01.009
Introduction
Nonribosomal peptides (NRPs) built a large pool of
biologically active natural compounds. The spectrum of
the clinical applications of NRPs is broad, for example
they are used as last resort antibiotics (daptomycin),
antitumor (bleomycin), or antifungal drugs or as immu-
nosuppressants (cyclosporin) (Figure 1a) [1]. This diverse
bioactivity can be explained by the way how nature
synthesizes these molecules. NRPs are produced in the
secondary metabolism of bacteria and fungi by the con-
secutive condensation of amino acids, which is achieved
by large multimodular enzymes, nonribosomal peptide
synthetases (NRPSs) [1,2]. Notably, this process is not
limited to the 20 proteinogenic amino acids. Some 500
different monomers, including nonproteinogenic amino
acids, fatty acids, and a-hydroxy acids, have been ident-
ified as building blocks for NRPs [3]. The nonproteino-
genic building blocks contribute to structural versatility of
NRPs and are likely to contribute substantially to the
observed biological activity. In brief, NRPSs are com-
Current Opinion in Structural Biology 2010, 20:234–240
posed of an array of distinct modular sections (Figure 1b),
each of which is responsible for the incorporation of one
defined monomer into the final peptide product. The
identity and order of a module in an assembly line
specifies: first, the sequence of monomer units activated
and incorporated; second, the chemistry that occurs at
each way station in the assembly line; and third, the
length and functionality of the product released from
the distal end of the assembly line (Figure 1b) [4].
The modules can be further divided into catalytic
domains. Three domains are ubiquitous in NRP synthesis
and essential for peptide elongation. The domains are
responsible for the activation of the amino acid (adenyla-
tion (A) domains), the propagation of the growing peptide
chain (thiolation or peptidyl carrier protein (PCP)
domains), and the condensation of the amino acids (con-
densation (C) domains). A fourth essential NRPS cataly-
tic unit associated with product release is the thioesterase
(TE) domain. The TE domain is located in the termin-
ation module and catalyzes peptide release by either
hydrolysis or macrocyclization [5].
During the last decade each domain structure was deter-
mined by either crystal or NMR structure elucidation
(Figure 1c); however, the dissected domain structures
only gave little knowledge of the mechanism underlying
the domain interaction during NRP synthesis [6].
Recently, different conformations and structural states
of PCP and A domains were observed. The implications
regarding the reaction cycles of PCP and A domains are
discussed herein. In addition, during the last three years,
it was possible to obtain high-resolution NMR and crystal
structures of didomains and even an X-ray structure of an
entire NRPS termination module, by trapping the most
flexible domain, the PCP domain in a certain state. This
review features the structural insights into modular NRP
synthesis by detailing the recent results in multidomain
structures. The possibility how these findings might assist
future NRPS re-engineering experiments as well as what
experiments remain to be done to fully understand the
mechanisms underlying NRP synthesis are discussed in
the last section of this article.
Reaction cycles of peptidyl carrier and
adenylation domains
Extensive NMR and X-ray structure elucidation exper-
iments provided insights into the PCP and A domain
reaction cycles and are depicted in Figure 2. In the first
step of the PCP cycle, the carrier (80 aa’s), which is
responsible for the transportation, propagation, and pres-
entation of the aminoacyl or peptidyl substrates of the
growing NRP chain, is primed post-translationally with its
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 Nonribosomal peptide synthetases Strieker, Tanovic and Marahiel 235

Figure 1

(a) Examples of nonribosomally assembled, clinically approved peptides. Next to the compound the names (first line), the trade names (second line),
and the biological activity are given (third line). (b) Simplified mechanism of nonribosomal peptide (NRP) synthesis. (1) The amino acid is activated as
aminoacyl-AMP by the adenylation domain. (2) Transfer of the amino acid onto the PCP domain. (3) Condensation of PCP-bound amino acids. (4)
Possibility of amino acid modifications, for example by epimerization domains. (5) Transesterification of the peptide chain from the terminal PCP onto
the TE domain. (6) TE catalyzed product release by either hydrolysis or macrocyclization. The number of modification domains and modules is very
variable. (c) Examples of single domain structures [20,26,45,46]. PDB accession codes are given in parenthesis.

40 -phospopantetheine (ppant) cofactor [7]. The priming
reaction, in which the ppant-arm is attached covalently to
a highly conserved serine residue (GGXS-motif), is car-
ried out by ppant-transferases such as the promiscuous
Bacillus subtilis ppant-transferase Sfp [8]. The PCP
domain exists in three different and converging confor-
mations: the apo (A), the A/H, and the holo (H) state. For
the apo-PCP, the A and A/H conformations coexist and in
the case of holo-PCP the presence of H and A/H states
were identified by NMR studies of an excised PCP
domain (TycC3–PCP) [9]. Thus, the common state for
apo and holo is the A/H state. It was shown that during the
priming reaction Sfp exclusively interacts with the A-state
to generate the ppant-primed PCP domain. Approxi-
mately 80% of coenzyme A (CoA), the precursor of the
ppant cofactor, is acetylated in bacteria [10], thus mis-
priming events during the Sfp-mediated PCP phospan-
tetheinylation occur quite regularly, which interrupt the
NRP assembly (Figure 2). These dead end misprimed
PCP species are repaired by the action of so-called type II
thioesterases (TEII) [11]. The NMR structure of the
TEII of the surfactin biosynthesis cluster has been elu-
cidated recently and gave valuable insights into the
mechanism of the crucial NRPS repair enzyme [12].
These studies revealed that the TEII interacts specifi-
cally with the misprimed PCP domain in its H state. In
addition, it was shown that only small molecules (e.g.
acetyl groups) are cleaved of in order to regenerate the

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236 Macromolecular assemblages

Figure 2

PCP and adenylation domain cycle. The apo-PCP domain (left half, green) is converted to the holo-form by a ppant-transferase (e.g. Sfp, left half, cyan)
by ppant attachment to the active site serine. Mispriming, for example with acetyl-ppant is repaired by the thioesterase type II (left half, orange). The
correctly primed holo-PCP domain interacts with the A domain cycle where it gets loaded with an amino acid. After translocation of the amino acid by
condensation, the holo-PCP can be loaded again. In the A domain cycle (right half), the small C-terminal subdomain (brown) of the A domain (red)
traverses several states. In an open conformation, the A domain is able to bind the amino acid and ATP. The adenylation intermediate then generates
the aminoacyl-AMP and pyrophosphate is cleaved off. The aminoacyl-AMP intermediate is protected from bulk solvent in a closed formation, which
likely facilitates transfer onto the PCP domain (thiolation).

thiol group of the ppant-arm of the holo-PCP. After holo-
PCP generation either by direct ppant-transfer or by
regeneration of misprimed PCPs, the cross-section be-
tween the PCP and the A domain cycle is reached and the
PCP is ready for amino acid loading (Figure 2).
In brief, before addressing the A domain cycle, some
background information on A domains: adenylation
domains are highly selective gatekeeper enzymes for
incoming monomeric NRP building blocks. Pioneering
work from the late 1990s on a large number of adenylation
domains, in which biochemical, structural, and phylo-
genic studies were combined, revealed the so-called
specificity-conferring code of A domains [13]. This code
is composed of 10 amino acids, which, by sequence
alignment with the first A domain structure PheA [14],
are responsible for substrate binding within the active site
of A domains. Subsequent bioinformatic studies allowed
the prediction of the substrate amino acids directly from
the primary sequence. Different online tools for A domain
specificity prediction are available [15,16] and might
facilitate the isolation of predicted NRPS products in
genome mining approaches [17,18]. Each A domain
comprises a large N-terminal core domain (Acore, 450
aa’s, Figure 2, illustrated in red) and a small C-terminal
subdomain (Asub, 100 aa’s, Figure 2, illustrated in
brown), which are connected by a small 5–10 residues
hinge. The core and subdomain organization of A
domains is highly conserved even in other adenylate
generating enzymes, such as acetyl-CoA synthase or fire-
fly luciferase (for a comprehensive review on adenylating
enzymes, see Gulick [19]). In a recent structure report of
DltA [20], a D-alanine activating adenylation enzyme,
extensive manual modeling of subdomain rearrangement
was performed to generate a potential cycle of the DltA
catalyzed reaction. This model cycle is also likely to be
applicable to NRPS A domains and hence presented in
Figure 2. The cycle starts with an open conformation of
the A domain (as observed for firefly luciferase [21]), in
which Asub is bend away from the active site. It is
proposed that this open structure is able to bind the
amino acid and ATP. Upon substrate docking (aa,
Mg2+, and ATP), an adenylating intermediate is formed,
as suggested from the crystal structure of DhbE in the
presence of 2,3-dihydroxybenzoate and AMP [22]. This
rearrangement of the subdomain is also supported by
steady-state and presteady-state kinetic experiments on
PheA in the presence of substrate and ATP [23]. Cleavage

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 Nonribosomal peptide synthetases Strieker, Tanovic and Marahiel 237

of the phosphoester bond with concomitant aminoacyl
adenylate formation and pyrophosphate release would
lead to a closed conformation, as observed in the DltA
structure [20]. In this state the aminoacyl-AMP is pro-
tected from bulk solvent. Then the activated amino acid
is transferred onto the PCP by a yet unknown mechanism,
representing the thioester-forming step. Release of the
loaded PCP domain, the thiolation product of the ade-
nylation domain complete reaction, would accompany a
rotation of Asub back to the open conformation (1408).
After restoring the PCP domain by translocation of the
amino acid by condensation, the combined thiolation–
adenylation cycle could start again (Figure 2).
PCP–C and PCP–TE didomain structures
In 2007 the X-ray structure of a PCP–C didomain [24]
from the third subunit of the tyrocidine synthetase [25]
(TycC5–PCP–TycC6–C) was determined at 1.8 Å resol-
ution (Figure 3a). During tyrocidine biosynthesis the
ultimate C domain (TycC6–C) catalyzes the last peptide
bond formation between the TycC6–PCP-bound Leu
and a nonapeptide bound to the penultimate carrier
domain (TycC5–PCP). It is essential that the involved
PCP domain is able to reach all domains necessary for
peptide elongation, that is upstream and downstream
condensation domains and the upstream A domain. Crys-
tals of the didomain enzyme were grown from hom-
ogenous apo-PCP containing protein solution. In the
crystal structure, the PCP domain (Figure 3a, green)
was found in a fold similar to the A/H state described
in the TycC3–PCP NMR studies [9]. The C domain has
V-shaped architecture as previously observed in the free-
standing C domain VibH [26] (Figure 1c). Within this V-
shaped structure the two equally big subdomains, which
belong to the chloramphenicol acetyltransferase fold
[27], are connected only by a small hinge region at the
bottom of the V and by a bridging loop in the middle,
which results in a canyon-like active site structure with
the catalytically active H224 (Figure 3a, shown in yellow)
in the middle. The V-shaped fold allows the two
upstream and downstream PCP domains to position
the condensation substrates at each opening (acceptor
and donor sites). In the didomain the PCP’s active site
serine (site of ppant attachment) is rotated away from the
C domain’s active site histidine by approximately 47 Å
(Figure 3a) [24]. The length of the ppant-arm (not pre-
sent is the structure) is 20 Å, that is in this orientation
the PCP–C interaction is unproductive. One could
speculate for possible explanation for this orientation:
first, the orientation is caused by crystal packing forces
leading to a nonphysiological architecture; second, the X-
ray snapshot could be an intermediate state between
catalytic cycles; third, it reflects a state in which the
PCP is oriented backwards to the upstream C domain
(not present in the structure). In the latter case, the PCP
domain must undergo enormous, yet not impossible,
rearrangements during one round of catalysis to serve
all essential domains. Until the structure of a C–A–PCP–
C multidomain has not been solved, these possibilities
remain speculative. A second didomain structure [28]
was released in 2008, which features the solution NMR
structure of the apo-peptidyl carrier protein–thioesterase
(PCP–TE) fragment of the enterobactin synthetase
EntF [29] NRPS subunit. To assure a homogeneous
apo-PCP–TE didomain the active site Ser 48 was
replaced by Ala (S48A, Figure 3b). This apo species
represents the physiological structure before CoA
addition. The S48A PCP active site is positioned
18 Å from the TE active site Ser 180, that is in reachable
distance of the 20 Å ppant-arm. The PCP and TE

Figure 3

Didomain structures. (a) X-ray structure of the PCP–C didomain [24] of the fifth PCP and sixth C domain of the tyrocidine NRPS subunit TycC. The
active site serine 43 (shown as orange spheres) of the PCP domain (green) is located 47 Å away from the active site histidine 224 (shown as yellow/blue
spheres) of the C domain (gray). The sites where the peptidyl substrate (from the PCP domain in the structure) is donated and where the growing chain
is accepted by the downstream PCP domain are labeled accordingly. (b) NMR structure of the PCP–TE didomain [28] of the termination module of
the enterobactin NRPS subunit EntF. The PCP active site serine was mutated to alanine (S48A). The distance between S48A and the active serine 180
of the TE domain is 18 Å. The catalytic triad of the TE domain is highlighted in spheres (H313–D207–S180). Interdomain linkers are colored in blue.

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238 Macromolecular assemblages

domains have a well-defined, but also flexible, relative
orientation with a primarily hydrophobic interface of
approximately 1300 Å2 [28]. This strong interaction
might explain the need to shuffle PCP–TE fragments
together in previous NRPS re-engineering efforts
[30,31].
Structure of a NRPS termination module
Recently the crystal structure of the four domains con-
taining termination module of the surfactin NRPS
(SrfA–C) was determined (Figure 4) [32]. Within the
SrfA–C structure, the individual domains show the same
folds as observed previously for the dissected domains
(Figure 1c) [33,34]. It was necessary to mutate the ppant
binding serine to alanine within the PCP domain to
obtain a uniform apo population of protein and to reduce
the mobility of domains during crystallization. The
structural core of the SrfA–C module is a compact
rectangular catalytic platform, built by the C domain
and the major part of the A domain (Acore) with both
active sites arrayed on the same site of the platform
leading to an interaction surface of more than 1600 Å2
[32]. The C–Acore interface can be seen as a stable
workbench similar to the KS-(M)AT-platform in the
condensing wing of the fatty acid synthase [35,36] or
the KS-AT-platform in type I polyketide synthases
[37,38]. In addition, the strong interaction between
the Acore and the C domain explains why these two
domains have to be shuffled together in NRPS re-engin-
eering approaches in order to create a functional assem-
bly line [39]. In this structure, the adenylation domain is
positioned in a conformation similar to the adenylate-
forming conformation (Figure 2). The C-terminal sub-
domain is rotated away from the N-terminal domain by
approximately 408 compared to the PheA structure [14],
resulting in a more open active site [32]. The PCP
domain and the Asub domain have little interaction with
the platform and thus can move to adopt the required
conformation during the catalytic cycle, as the active
sites of the A and the C domains are separated by
approximately 60 Å. In the observed state for the PCP
domain, it is impossible to cover this distance, as the
ppant-arm is only 20 Å in length. The regions, which
could be reached in the SrfA–C structure by the ppant
cofactor are covered by a gray sphere in Figure 4. The
PCP domain of the SrfA–C protein is positioned to
interact productively with the upstream condensation
domain. The domain is likely to be positioned in the
acceptor site of the C domain, as the catalytic center of
the PCP domain is only 16 Å (Figure 4) from the active
site histidine of the C domain. The PCP active site
serine is not positioned, where it could donate the
pantetheine arm to either the adenylation domain or
the thioesterase domain [32]. Therefore large struc-
tural rearrangements are mandatory during a full cata-
lytic cycle.

Figure 4

Back view or acceptor site view of the termination module of the surfactin NRPS subunit SrfA–C. The 20 Å radius, which is the reachable region of the
carrier-arm, around the ppant-attachment site of the PCP domain (S1003A) is depicted as a gray sphere. The PCP domain is well positioned to interact
with the H144 active site of the C domain. The active sites of the TE (S1117) and the A domain substrate leucine cannot be reached. The active site of
each domain is shown in spheres. Linker regions are shown in blue, the domains are arranged and colored corresponding to the schematic illustrations
in upper left corner.

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 Nonribosomal peptide synthetases Strieker, Tanovic and Marahiel 239
Conclusions
Although remarkable progress regarding the revelation of
the mechanisms underlying NRP synthesis has been
made during the last decade, we are still far away from
understanding the whole picture. The highlighted recent
work presented in this review, gave valuable insights into
certain parts of NRP synthesis especially in the field of
carrier protein [9] and adenylation domain dynamics
[20]. Furthermore, important protein–protein interaction
surfaces were determined as in the case of the PCP–TE
[28] and the C–Acore-platform [32]. These interactions
explain previously observed biocombinatorial problems
regarding NRPS re-engineering efforts [30] and may
guide future directions. In addition, the structure of
the NRPS termination module [32] of the surfactin
synthetase SrfA–C provides first structural insights into
the architecture of an entire NRPS module and gives
hints how every essential domain could be reached by
conformational changes and rearrangements of the PCP
domain. However, additional X-ray structure elucidation,
for example of multimodular NRPS would be helpful as
well as of modules containing native holo-PCP domains,
that is without mutated ppant-attachment site. This
should provide a better understanding of intramodular
and intermodular interactions. These could be achieved,
for example by the usage of nonhydrolyzable CoA-ana-
logs [40,41], which can be loaded artificially on the PCP
domain by Sfp [8] and might freeze the PCP domain in
the donor site of the C domain or by interdomain cross-
linking [42]. In addition the flexibility of the A domain
could be restricted by mutating certain residues in the
hinge region as suggested by Gulick [19]. As exemplified
by the potentially nonphysiological X-ray PCP–C struc-
ture [24], structural dynamics have to be determined in
future. These can be measured either by NMR studies on
single or didomains, as it becomes easier to get NMR
structures of enzymes in the 50 kDa range (size of C and
A domains), e.g. by using stereo-array isotope labeling
(SAIL) [43], or by small angle X-ray scattering [44] for the
entire assembly line.
Acknowledgements
We gratefully acknowledge the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie for financial support.
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