Biomimetic catalysis of intermodular aminoacyl transfer

Article abstract of DOI:10.1021/ja067124h

Intermodular aminoacyl transfer is the fundamental bond-forming reaction in the biosynthesis of polypeptides by ribosomes and nonribosomal peptide synthetases (NRPS). Here we report the design and functional characterizations of short synthetic α-helical

Full text of DOI:10.1021/ja067124h

Published on Web 01/05/2007
Biomimetic Catalysis of Intermodular Aminoacyl Transfer
Keith M. Wilcoxen, Luke J. Leman, Dana A. Weinberger, Zheng-Zheng Huang, and M. Reza Ghadiri*
Departments of Chemistry and Molecular Biology and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received October 4, 2006; E-mail: ghadiri@scripps.edu
Intermodular aminoacyl transfer is the fundamental bond forming
reaction in the biosynthesis of polypeptides by ribosomes and
nonribosomal peptide synthetases (NRPS).1 Here we report the
design and functional characterizations of short R-helical peptides
that mimic the aminoacyl loading and intermodular aminoacyl
transfer steps of NRPS with aminolysis rate enhancements in neutral
aqueous solutions of up to 5400-fold (kcat/kuncat). The catalysts
operate as noncovalently associated assemblies with composite
active sites fashioned at the interface between helical subunits.
Following substrate loading at the active site cysteine, the juxta-
position of the resulting aminoacyl thiolester and the amine of the
acyl acceptor moiety gives rise to high effective concentrations (up
to 54 M) that facilitate interhelical aminoacyl transfer. Moreover,
studies based on homo- and heteromeric assemblies, active site
substitutions, kinetic analysis, and reaction modeling indicate that
the supramolecular catalysts exhibit some basic characteristics of
natural enzymes, including precise positioning and pKa modulation
of active site residues, covalent catalysis, and product turnover.2,3
In the present study, our primary objective was to mimic the
two fundamental chemical steps of NRPS: aminoacyl substrate
anchoring and intermodular aminoacyl transfer. To bring about
efficient acyl transfer, we sought to exploit primarily principles of
catalysis by approximation4 using 26-residue coiled-coil peptides5
to assemble noncovalently the aminoacyl donor and acceptor
moieties into productive complexes (Figure 1 and Figure S1,
Supporting Information). Because parallel coiled-coil homotetramers
are pseudo-4-fold symmetric, each complex contains four putative
active sites (Figure 1a). We designed and evaluated three active
site variations consisting of Cys for substrate loading and one of
the following acyl acceptors: a lysine residue (type-I), an aminoacyl
ester tethered at a serine residue (type-II), and a second aminoacyl
thiolester tethered at a cysteine residue (type-III) (Figure 1b).
We initially evaluated type-I active sites (peptides 1-4) using
100-fold excess Cbz-protected N-acetylcysteamine glycyl thiolester
as substrate. Under the neutral aqueous conditions, 1 underwent
substrate loading at the active site Cys to form 1a, subsequent
interhelical aminoacyl transfer to produce 1b, and reloading to yield
product 1c (Figures 2, S3, Table 1). Increases in the aminolysis
rate of 5400-fold were observed for 1 relative to the background
aminoacylation of Lys in control tripeptide 7. The interhelical mode
of acyl transfer was supported by mixing purified 1c with 1, which
resulted in rapid aminoacyl transfer to yield approximately 2 equiv
of 1b (see Figure S2 for details and an additional proof using labeled
peptides). The rate of aminoacyl transfer for 1 was reduced by
>300-fold in 6 M Gnd‚HCl as would be expected owing to the
partial unfolding of the coiled coil. Intermolecular rates of lysine
aminoacylation in the context of the folded scaffold were determined
by assaying 8 and 9, which lack an active site Cys residue.
Interestingly, the Lys residues in folded peptides 8 and 9 were
acylated 50- and 10-fold faster than Lys in unstructured tripeptide
7, respectively. Preliminary results suggest that this enhanced
Figure 1. Representations of the coiled-coil scaffold and the composite
aminoacyl transfer active sites. (a) (left) Helical wheel diagram of the
homotetrameric coiled-coil illustrating the four symmetry-related active sites
juxtaposing an aminoacyl donor (cysteine for covalent substrate anchoring
via transthiolesterification), an aminoacyl acceptor (amine from lysine or a
covalently tethered amino acid), and X1 and X2 residues potentially providing
electrostatic or general acid-base catalysis. (right) The 2.17 Å crystal
structure of a designed homotetramer6 illustrating the juxtaposition of the
putative active site residues. (b) Schematic illustration of the three active
site designs, highlighting aminoacyl transfer from an aminoacyl-donor to
the -acceptor moiety located on an adjacent helix. For clarity, only one of
the four symmetry-related active sites is shown (boxed region in the helical
wheel diagram). For peptide sequences see Table S1.
reactivity is due to a depression of the Lys active site pKa.2a,f
Substrate generality was assessed using a representative set of
L-aminoacyl thiolesters (Table S2). Although substitutions of the
R-substituent had little effect on the aminoacyl transfer rates (k2
varied by ∼3-fold), side chain â-substitution resulted in 7 to 13-
fold slower aminoacyl loading rates. To probe the influence of active
site histidines (X1 and X2) on the aminoacyl loading and transfer
steps, 2-4 were employed in which either or both residues were
substituted with alanine. While reductions in the acyl transfer rate
of up to 13-fold resulted, the observed acyl transfer efficiency of
4 discounts the possibility of acylimidazolyl intermediates in the
reaction.2c,3
The potential for aminoacyl transfer between two proximally
tethered amino acids was assessed using type-II and type-III active
site designs (Figure 1b). Encouragingly, 5 and 6 having a serine-
anchored aminoacyl ester as the acyl acceptor (type-II) underwent
substrate loading and aminoacyl transfer with rates similar to the
analogous reaction for 1 (Figure S5, Table 1), although the reduced
transfer rate for 6 likely results from the increased steric influence
of the acyl acceptor R-substituent. The possibility of aminoacyl
transfer in the type-III active site was studied using preloaded
aminoacyl donor (10a) and acceptor (11a, R ) H, Me) modules
(Figure 2). In this system Cys8 in 10a and Cys13 in 11a are disabled
by Acm side-chain protection. However, combining 10a and 11a
allows the formation of heterotetrameric assemblies which brings
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10.1021/ja067124h CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. (a) Schematic representation of catalytic aminoacyl loading and
transfer cycles based on heterotetrameric assemblies (type-I active site).
Colored circles represent R-helices for the donor peptide 12 (pink) and
acceptor peptide 9 (blue). For clarity, we depict only the statistically most
predominant catalytically competent heterotetrameric coiled-coils. (b)
Aminoacylation of 9 (∼360 µM) in reactions containing catalyst 12 at
varying concentrations. The curves shown represent best fits of the data to
a minimal reaction model, yielding rate constants of k1 ) 1.4 × 10-3 sec-1
and k2 ) 11.0 × 10-4 sec-1. The catalyst shows multiple turnovers
(∼1 turnover per hour for the first 20 h in the 3 mol % reaction).
self-assembling peptides. However, the hallmark of NRPS lies in
their ability based on the logic of their domain organization to
instruct the formation of specific peptide sequences.1 It remains to
be seen whether the supramolecular approach described here can
be further advanced toward programmed peptide synthesis by
exploiting the sequence-dependent selective coiled-coil assembly
recently demonstrated in the design of complex networks.8
Figure 2. Reactions schemes and product formation in time for type-I and
-III active sites. For type-I, reactions contained ∼100 µM peptide, 10 mM
Cbz-Gly-SNAC, 10 mM triscarboxyethyl phosphine (TCEP) as reducing
agent, 285 mM Hepes pH 7.0, and ∼200 µM acetamidobenzoic acid (Aba)
as internal concentration standard. Curve fits shown are from reaction
modeling using SIMFIT.7 Type-III reactions were initiated under similar
conditions by combining 10a (∼70 mM) with 11a (∼560 mM).
Acknowledgment. We thank NIH (Grant GM57690) for
financial support and NSF for a predoctoral fellowship (L.J.L.).
Table 1. Aminoacyl Loading and Transfer Rate Constantsa
Supporting Information Available: Experimental details, bio-
physical characterizations, Tables S1-S2, and Figures S1-S5. This
material is available free of charge via the Internet at http://pubs.acs.org.
1
1
peptideb
active site residues
k1 (10-3 sec-
)
k2 (10-4 sec-
)
1
2
3
4
5
6
1
7
8
9
K_HH_C
K_AH_C
K_HA_C
K_AA_C
1.3
1.0
1.3
3.4
1.3
0.9
0.5
e
9.2
1.3
0.7
0.5
9.1
0.4
0.03
0.0017
0.09
0.02
References
SGly_HH_Cc
SAla_HH_Cc
K_HH_Cd
Aba-SKL-CO2H
K_HH_S
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e
e
K_AH_S
a Reaction conditions are as described in Figure 2. b See Table S1 for
peptide sequences. c Aminoacyl-esterified Ser residue (type-II). d In 6 M
Gnd‚HCl, pH 7.0. e Transthiolesterification not possible.
together aminoacyl-donor and -acceptor moieties to create com-
petent active sites (Figure 2c, S4). Indeed, reaction mixtures
containing 10a and 11a resulted in aminoacyl transfer with rates
similar to those observed for type-I and -II active sites (Figure 2),
suggesting fast helix exchange followed by efficient intermodular
aminoacyl transfer.
We next examined the viability of catalytic turnover by exploiting
heterotetrameric peptide assemblies. Combining 9, which contains
an active site aminoacyl-acceptor (Lys) but no -donor (Cys), with
12, having an aminoacyl-donor but no -acceptor, makes possible
the formation of heterotetrameric assemblies in which Cys and Lys
residues are brought into proximity to form composite type-I active
sites (Figure 3a). As expected, the observed rates of aminoacylation
depended markedly on the concentration of 12 present (Figure 3b),
reflecting catalyst participation in the reaction. Multiple product
turnovers were observed, suggesting that helix subunit exchange
rates are faster than the rate of intermodular aminoacyl transfer.
The studies reported here establish that two fundamental steps
of NRPS can be effectively mimicked by appropriately designed
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