Reprogramming nonribosomal peptide synthetases for "clickable" amino acids

Article abstract of DOI:10.1002/anie.201405281

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan-to-serine mutation in phenylalanine-specific NRPS adenylation domains enables the efficient activat

Full text of DOI:10.1002/anie.201405281

Angewandte
Chemie
DOI: 10.1002/anie.201405281
Biosynthetic Engineering Very Important Paper
Reprogramming Nonribosomal Peptide Synthetases for “Clickable”
Amino Acids**
Hajo Kries, Rudolf Wachtel, Anja Pabst, Benedikt Wanner, David Niquille, and Donald Hilvert*
Abstract: Nonribosomal peptide synthetases (NRPSs) are
multifunctional enzymes that produce a wide array of bioactive
peptides. Here we show that a single tryptophan-to-serine
mutation in phenylalanine-specific NRPS adenylation
domains enables the efficient activation of non-natural aro-
matic amino acids functionalized with azide and alkyne
groups. The resulting 105-fold switch in substrate specificity
was achieved without appreciable loss of catalytic efficiency.
Moreover, the effective communication of the modified
A domains with downstream modules in dipeptide synthetases
permitted incorporation of O-propargyl-l-tyrosine into dike-
topiperazines both in vitro and in vivo, even in the presence of
competing phenylalanine. Because azides and alkynes readily
undergo bioorthogonal click reactions, reprogramming NRPSs
to accept non-natural amino acids that contain these groups
provides a potentially powerful means of isolating, labeling,
and modifying biologically active peptides.
Even complex, drug-related alkaloid or polyketide natural
products can be endowed with “clickable” alkyne groups,[5]
thereby facilitating biological studies and the search for new
drug candidates.[6]
Nonribosomal peptides (NRPs), an important source of
antibiotics and other biologically active agents, comprise
a particularly diverse class of natural products. They are
produced by mega-enzymes, called nonribosomal peptide
synthetases (NRPSs), that function as biosynthetic assembly
lines (Figure 1).[7] NRPSs consist of dedicated protein mod-
M
ethods for chemoselectively labeling biomolecules have
become indispensible in modern chemical biology.[1] Bioor-
thogonal reactions, such as copper(I)-catalyzed and strain-
promoted Huisgen cyclizations between alkynes and azides,
also known as “click” reactions,[2] are particularly useful in
this regard. In addition to being selective and high-yielding,
such reactions tolerate a broad range of functional groups.
They are consequently widely used to isolate, visualize, and
otherwise modify individual molecules in complex biological
samples.
Figure 1. Nonribosomal synthesis of the antibiotic gramicidin S.
A=adenylation domain; T=thiolation domain; E=epimerization
domain; C=condensation domain; TE=thioesterase domain.[7] The
phenylalanine residue encoded by A domain GrsAA in the first module
is highlighted in red.
Diverse strategies have been developed to equip biomol-
ecules with alkyne and azide functionalities. For example,
codon reassignment and nonsense suppression enable effi-
cient ribosomal production of proteins containing amino acids
with azide or alkyne side chains.[3] Click building blocks have
also been incorporated into DNA and cell-surface glycans.[4]
ules, strung together like beads on a string, which are
responsible for activating the amino acid building blocks
and incorporating them into the growing peptide chain. The
number, nature, and order of the individual modules deter-
mine the length and composition of the final natural product.
Such modularity has fueled efforts to produce novel bioactive
peptides by combinatorial biosynthesis, for example by
engineering, exchanging, or concatenating individual NRPS
domains or entire modules.[8]
[*] Dr. H. Kries, R. Wachtel, A. Pabst, B. Wanner, D. Niquille,
Prof. Dr. D. Hilvert
Laboratory of Organic Chemistry, ETH Zꢀrich
8093 Zꢀrich (Switzerland)
Although alkyne-functionalized chemical probes have
been used to assay the substrate specificities of NRPS
adenylation (A) domains,[9] a general method for click
functionalization of NRPs does not exist. As A domains
dictate molecular recognition in these systems, they could
conceivably be adapted for this purpose. Indeed, sequence
patterns deduced by comparisons of homologous active sites
have proven useful for predicting as well as altering A domain
specificity.[8b,10] Some conservative changes in substrate pref-
erence, such as l-Asp to l-Asn or 3-methyl-Glu to 3-methyl-
Gln, are relatively easy to achieve by targeted mutagenesis of
E-mail: hilvert@org.chem.ethz.ch
[**] We thank Prof. Mohamed A. Marahiel (Philipps Universitꢁt Mar-
burg) for supplying strain HM0079 and plasmids pSU18_tycA and
pTrc99a_tycB1, and Prof. Hans-Martin Fischer (ETH Zꢀrich) for
assistance with radiochemical experiments. We are also grateful to
Prof. Chaitan Khosla (Stanford University) for valuable discussions.
This work was supported by the Schweizerischer Nationalfond and
the ETH Zꢀrich. H.K. was supported by fellowships from the
Stipendienfonds der Schweizer Chemischen Industrie and the
Studienstiftung des deutschen Volkes.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201405281.
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residues lining the substrate binding pocket.[8b,10b,d] However,
redesigning A domains for recognition of non-cognate amino
acids that exhibit larger differences in size or charge is
generally more difficult, and adenylation efficiency often
drops substantially.[10e,f] These challenges notwithstanding, we
show here that only minimal changes are needed to reengi-
neer phenylalanine-specific A domains for the efficient
incorporation of aromatic amino acids containing alkyne
and azide functional groups into NRPs.
To expand the recognition properties of GrsAA, a phenyl-
alanine-specific A domain (PheA) from gramicidin S synthe-
tase (Figure 1),[7b,11] eight active-site residues known to
correlate with substrate specificity[10a,b] were chosen for
cassette mutagenesis (Figure 2A,B). The resulting single-
mutant libraries were individually screened against the twenty
Table 1: Catalytic parameters of the adenylation reaction catalyzed by
GrsAA and GrsAA-W239S.[a]
Variant
Substrate
kcat
kcat/Km
Speci- Specificity
[minÀ1
]
[mmÀ1 minÀ1 ficity[b] switch[c]
]
wt
wt
wt
W239S
W239S
l-Phe
p-azido-l-Phe
O-propargyl-l-Tyr
l-Tyr
300
200
30
230
200
190
25000
25
2
600
9000
7000
1
1
–
–
10À3
10À5
3.2
47
800
5ꢂ104
5ꢂ105
p-azido-l-Phe
W239S O-propargyl-l-Tyr
37
[a] Catalytic parameters were determined by a pyrophosphate exchange
assay.[10e,12] For additional kinetic data and experimental errors see
Table S1. [b] Specificity: kcat/Km (target substrate)/kcat/Km (l-Phe). [c] Spe-
cificity switch: (specificity)mut/(specificity)wt for the indicated amino acid.
Simple modeling suggested that the W239S mutation
opens up a cavity large enough to accommodate amino acids
even bulkier than tyrosine (Figure 2C and D). Although l-
Trp was inefficiently adenylated (kcat/Km = 36 mmÀ1 minÀ1),
phenylalanine derivatives bearing bulky para substituents
were excellent substrates. Adenylation rates increased with
the size of the para-substituent in the order H < OH < Cl <
OMe < OEt (Figure 2E; Table S1). The p-ethoxy substituent
afforded remarkably high activity (kcat/Km = 50000
mmÀ1 minÀ1), greater than that of wt GrsAA with l-Phe
(kcat/Km=25000 mmÀ1 minÀ1). The variations in adenylation
activity manifest in these kcat/Km values largely reflect differ-
ences in Km for the different amino acids. Under saturating
conditions at high substrate concentration, all turnover
numbers, including those with l-Trp, were in the range of
GrsAA with l-Phe, i.e. 102 to 103 minÀ1 (Table S1).
The capacious GrsAA-W239S binding site also supported
efficient activation of click amino acids containing azide and
alkyne functionality. Thus, p-azido-l-Phe and O-propargyl-l-
Tyr, which are poorly processed by wt GrsAA, serve as
excellent substrates for the mutant PheA domain (Fig-
ure 2E). For example, GrsAA-W239S adenylated O-prop-
argyl-l-Tyr 10 times more rapidly than l-Tyr, the best
naturally occurring amino acid substrate, and 40 times faster
than l-Phe. These changes, corresponding to a 5 ꢀ 105-fold
switch in enzyme specificity (Table 1), were achieved with
little loss in catalytic efficiency, judging from the steady-state
parameters for the mutant enzyme with the non-natural
substrate (kcat = 190 minÀ1 and kcat/Km = 7000 mmÀ1 minÀ1)
relative to wt GrsAA with l-Phe (kcat = 300 minÀ1 and
kcat/Km = 25000 mmÀ1 minÀ1).
Figure 2. Engineering the GrsAA binding pocket. A) Crystal structure of
GrsAA (PDB code 1AMU)[11] with bound l-Phe (green carbon atoms)
and adenosine monophosphate (yellow carbon atoms). B) Residues
lining the l-Phe binding pocket are shown as stick models. The amino
acids in cyan and magenta (Ala236, Trp239, Thr278, Ile299, Ala301,
Ala322, Ile330, and Cys331) were individually subjected to cassette
mutagenesis; Cys331 is hidden behind the substrate. Asp235 and
Lys517 (gray), important for catalysis, were not mutated. Adenosine
monophosphate was omitted for clarity. C) Cut-away view of the
binding pocket in the plane of the phenyl ring. D) PyMOL model of the
W239S variant. E) Adenylation kinetics of GrsAA (gray) and GrsAA-
W239S (green) with a range of aromatic amino acid substrates,
including the click amino acids p-azido-l-Phe and O-propargyl-l-Tyr.
See Table 1 and Table S1 in the Supporting Information for details.
proteinogenic amino acids using a microtiter-plate-based
pyrophosphate exchange assay for adenylation activity.[10e,12]
Replacing Trp239, located at the bottom of the PheA
recognition pocket, with smaller amino acids afforded prom-
ising changes in substrate specificity. Substituting tryptophan
with serine (W239S), for example, resulted in an approxi-
mately three times higher preference for l-Tyr over l-Phe,
which corresponds to an 800-fold switch in specificity relative
to wild-type (wt) GrsAA. The kcat/Km value for l-Tyr
activation by this variant is 600 mmÀ1 minÀ1, only 40 times
below the kcat/Km determined for l-Phe with the wild-type
enzyme (Table 1).
Adenylation activity alone is insufficient for nonriboso-
mal peptide synthesis with unnatural building blocks; per-
missivity of downstream domains is also essential. To test
whether the modified GrsAA domain can communicate with
other domains and modules, we employed a truncated GrsA/
GrsB1 dipeptide synthetase excised from the gramicidin S
NRPS (Figure 3).[13,14] In addition to the PheA domain, the
complete GrsA initiation module contains a thiolation (T)
and an epimerase (E) domain, whereas GrsB1 is a truncated
elongation module containing a proline-specific A domain,
a T domain, and a condensation (C) domain. The wild-type
dipeptide synthetase loads l-Phe and l-Pro onto their
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competent for peptide chain
elongation. Moreover, the
specificity observed for the
isolated domain was main-
tained in the context of the
model synthetase. When
both amino acids were pres-
ent in equal amounts under
competitive conditions, the
mutant synthetase preferen-
tially incorporated O-prop-
argyl-l-Tyr into the DKP
product by
roughly 70:1, exceeding the
adenylation specificity
a factor of
observed with the isolated
GrsAA-W239S domain.
As Trp239 is a common
residue in PheA domains
(Table S3), the specificity
switch achieved by mutating
this residue is potentially
general. The portability of
the W239S substitution was
tested with the homologous
TycA protein from tyroci-
dine synthetase,[17] which
shares 62% sequence iden-
tity with GrsA. The result-
ing variant displayed high
turnover with O-propargyl-
Figure 3. Nonribosomal synthesis of propargylated DKP. A) Mechanism of nonribosomal DKP formation by
the truncated NRPSs. B–E) Kinetics of DKP formation from 500 mm l-Pro, l-Phe, and O-propargyl-l-Tyr as
competing substrates were determined with different dimodular synthetases: B) GrsA/GrsB1, C) GrsA-
W239S/GrsB1, D) TycA/TycB1, and E) TycA-W239S/TycB1. kobs values are summarized in Table S2. Concen-
trations of d-Phe-l-Pro DKP (black circles), O-propargyl-l-Tyr-l-Pro DKP (open cyan squares), and
O-propargyl-d-Tyr-l-Pro DKP (cyan squares) were determined by UPLC. F) UPLC traces of DKP standards (1:
O-propargyl-d-Tyr-l-Pro DKP, 2: O-propargyl-l-Tyr-l-Pro DKP, 3: d-Phe-l-Pro DKP) and propargyl DKP pro-
duced in vivo (4: reaction t=0 h, 5: t=24 h, 6: purified product). The peak at 3.9 minutes corresponds to
O-propargyl-l-Tyr. Traces were normalized to the height of the DKP peak.
respective T domains and, following epimerization of phenyl-
alanine and peptide bond formation at the active site of the
C domain, affords a d-Phe-l-Pro thioester that is spontane-
ously released as a d-Phe-l-Pro diketopiperazine (DKP;
Figure 3A).[13,14]
l-Tyr in the pyrophosphate exchange assay (kcat
=
280 minÀ1), but its catalytic efficiency, judged by the kcat/Km
parameter (850 mmÀ1 minÀ1), was somewhat lower than that
with GrsAA-W239S (Table S1). Nevertheless, TycA-W239S
exhibited an eight-fold preference for O-propargyl-l-Tyr over
l-Phe. Moreover, it successfully communicated with the
downstream proline-specific TycB1 module to produce prop-
argylated DKP, even in the presence of competing l-Phe
(Figure 3D,E). The initial rate of DKP formation was four
times faster than for GrsA-W239S/GrsB1 and only 1.7 times
slower than the natural reaction sequence catalyzed by wt
TycA/TycB1 (Table S2). In this case, the unnatural substrate
was properly epimerized by the TycA E domain prior to chain
elongation to give propargylated DKP in a d,l/l,l ratio of
95:5, similar to that observed for the wild-type dimodule.[16]
Intracellular peptide biosynthesis constitutes the most
stringent test for incorporation of non-natural building blocks
into NRPs. Despite potential interference from competing
endogenous amino acids, the activity and selectivity of the
engineered TycA-W239S/TycB1 synthetase enabled efficient
in vivo production of propargylated peptide. Following
24 hour incubation with O-propargyl-l-Tyr (500 mm) and l-
Pro (500 mm) at 378C, propargylated DKP was isolated from
a bacterial culture (1 L) co-expressing TycA-W239S and
TycB1. Purification of the organic extract by HPLC afforded
10 mg of the d,l-configured product. The biosynthetic DKP
was identical to a chemically synthesized standard, as
corroborated by its UPLC retention time (Figure 3F) and
Phosphopantetheinylated GrsA-W239S and GrsB1 mod-
ules were produced in E. coli strain HM0079, which endoge-
nously expresses phosphopantethein transferase Sfp.[15] The
proteins were purified by NiNTA affinity chromatography
and assayed for DKP synthesis. In the presence of equimolar
amounts of O-propargyl-l-Tyr, l-Phe, and l-Pro, the GrsA-
W239S/GrsB1 synthetase generated propargylated DKP only
4.5 times more slowly than wt GrsA/GrsB1 produced d-Phe-
l-Pro DKP under identical conditions (Figure 3B,C). How-
ever, in contrast to the 98:2 preference for the d,l over the l,l
product exhibited by the wild-type synthetase,[16] the variant
afforded predominantly l,l-configured DKP. Only around
10% of the d,l product isomer was observed, indicating
inefficient epimerization of O-propargyl-l-Tyr by the initia-
tion module. The propargyloxy substituent may interfere with
binding to the epimerase or, alternatively, override the
inherent stereochemical preference of the downstream C do-
main for a d-configured aminoacyl thioester.[13] Damage to
the E domain can be ruled out by the fact that, in the absence
of O-propargyl-l-Tyr, GrsA-W239S/GrsB1 converts l-Phe
and l-Pro quantitatively to the corresponding epimerized
product within one day. Despite only partial epimerization,
these results clearly demonstrate that the mutant A domain is
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1H NMR spectrum (Figure S1). Its yield also compared
favorably with the 9 mglÀ1 of d-Phe-l-Pro DKP produced
by the wild-type enzyme under similar conditions.[15] As d-
Phe-l-Pro DKP was barely detectable in the sample, the
intracellular concentrations of O-propargyl-l-Tyr were evi-
dently high enough to outcompete endogenous l-Phe.
Efficient incorporation of this non-standard amino acid into
an NRP provides a potentially general means to attach
fluorescent labels, simplify purification, and modulate bioac-
tivity through postsynthetic click functionalization.
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The W239S mutation is a strikingly non-invasive, yet
powerful way to repurpose PheA domains. This single active
site substitution creates an enlarged binding pocket that
accommodates the unnatural amino acid O-propargyl-l-Tyr
(Km = 30 mm) without compromising catalysis. Both the activ-
ity and selectivity of the mutant for the new substrate are
almost on par with the wild-type enzyme. Importantly, too,
the downstream thioesterification, epimerization (in one
case), and condensation steps readily accommodate the
modified substrate. Achieving efficient activation of a click-
able amino acid in two homologous synthetases highlights the
potential generality of this strategy. Similar reprogramming of
other synthetases should be possible, as many NRPSs share
the phenylalanine recognition motif utilized by GrsAA and
TycAA. Synthetases that produce the natural products thax-
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Received: May 15, 2014
Published online: July 31, 2014
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Keywords: adenylation domain · mutagenesis · click chemistry ·
diketopiperazine · nonribosomal peptide
.
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