A minimalistic hydrolase based on co-assembled cyclic dipeptides

Article abstract of DOI:10.1039/c9ob02198a

The self-assembly of small peptides into larger aggregates is an important process for the fundamental understanding of abiogenesis. In this article we demonstrate that blends of cyclic dipeptides (2,5-diketopiperazines-DKPs) bearing either histidine or c

Full text of DOI:10.1039/c9ob02198a

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A minimalistic hydrolase based on co-assembled
cyclic dipeptides†
Alexander J. Kleinsmanna and Boris J. Nachtsheim
Cite this: Org. Biomol. Chem., 2020,
18, 102
b
*
The self-assembly of small peptides into larger aggregates is an important process for the fundamental
understanding of abiogenesis. In this article we demonstrate that blends of cyclic dipeptides (2,5-diketopi-
perazines – DKPs) bearing either histidine or cysteine in combination with a lipophilic amino acid form highly
stable aggregates in aqueous solution with esterase-like activity. We demonstrate that the catalytic activity is
based on an intermolecular cooperative behavior between histidine and cysteine. A high control of the mole-
cular arrangement of the peptide assemblies was gained by C–H-π interactions between Phe and Leu or Val
sidechains, resulting in a significant increase in catalytic activity. These interactions were strongly supported by
Hartree–Fock calculations and finally confirmed via 1H-NMR HRMAS NOE spectroscopy.
Received 10th October 2019,
Accepted 25th November 2019
DOI: 10.1039/c9ob02198a
rsc.li/obc
of other amino acid-derived hybrids.9,10 The relevance of these
approaches in abiogenesis is questionable due to the artificial
Introduction
The transition of simple small molecular building blocks, in nature of the underlying molecular building blocks.
particular fatty-, amino- and nucleic acids, into self-replicating In this regard, cyclic dipeptides (2,5-diketopiperazines –
systems with an autonomous metabolism is the critical step DKPs) are observed frequently as undesired side-products
for the emergence of the first living cells with a minimalistic during peptide formation and under prebiotic conditions,11 in
genotype and phenotype.1 The initial manifestation of small particular as degradation products of small oligopeptides.12
peptides as enzyme precursors that could have provided They form spontaneously upon thermal treatment of linear
important catalytic properties for autonomous self-replicating dipeptides in the solid state.13 In addition, they have been
systems is still underexplored.2,3 Here, self-assembly processes found on the Yamato-791198 and Murchison carbonaceous
that form higher ordered aggregates from spontaneously chondrites.14 We recently demonstrated that a variety of Phe-
formed
H-bonding interactions is believed to be an important initial solutions.15 Their self-aggregation is the result of strong
step.2,4a–d
H-bonding interactions between the cyclic amides and
small
oligopeptides
through
intermolecular containing DKPs form highly stable aggregates in aqueous
Artificial enzymes, in particular esterases based on the self- additional π–π– or C–H-π-interactions between the Phe side-
assembly of short oligopeptides have been described fre- chains. This was further revealed by fundamental investi-
quently.5 Gazit and co-workers recently demonstrated that gations of Govindaraju and co-workers on cyclic dipeptides
even single amino acids, in particular phenylalanine, in the containing Phe- or PhG side chains or Fmoc-protected lysine-
presence of zinc can form supramolecular amyloid-like struc- derived DKPs.16,17 Intermolecular hydrogen bond patterns
tures that render a potent esterase activity.6 However, usually, have also been observed in solid state structures of DKPs, for
lipophilic tripeptides, amphiphilic oligopeptides or amyloid- example by Marchesan and co-workers very recently.18 For
forming peptides are necessary to generate self-assembled proving the relevance of DKPs in the context of abiogenesis,
nanostructures with esterase-like activity.7 Catalytically active their catalytic properties must be elucidated. So far this has
aggregates can also be formed based on artificial dendrimers, only been demonstrated by Lipton and co-workers in solution
by fixation of a peptide onto nanoparticles,8 or the generation for the asymmetric Strecker reaction as well as in epoxidations
in multiphase systems as shown by Voyer and coworkers.19,20
Presuming their high tendency to aggregate in water into a
defined molecular arrangement, we proposed that simple
aInstitut für Organische Chemie Universität Tübingen, Auf der Morgenstelle 18,
72076 Tübingen, Germany
blends of two DKPs composed of proteinogenic α-amino acids
bInstitut für Organische und Analytische Chemie Universität Bremen, Leobener
with lipophilic side chains and differing “functional” side
Straße 7, 28359 Bremen, Germany. E-mail: nachtsheim@uni-bremen.de
chains should render enzyme-like catalytic activity in the co-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
assembled state through intermolecular cooperative effects.
c9ob02198a
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cycle in pure water at concentrations between 80 and 106 mM.
SEM and TEM images showed the appearance of nanofibers
Results and discussion
To verify this working hypothesis, we generated a minimalistic with varying average diameters ([1 + 2]: 12.3 nm, [1 + 3]:
hydrolase mimic (Fig. 1a). 32.6 nm and [1 + 4]: 21.4 nm) (for detailed analysis of repre-
In the catalytically active side of hydrolases, imidazoles of sentative SEM-images see ESI†).
His-residues are in close proximity to Ser, Cys or Asp side-
To investigate esterase-like activity of the co-aggregates, the
chains as the structural basis for catalytic dyads or triades. To hydrolysis of sodium 4-acetoxy-3-nitrobenzenesulfonate (ANBS,
the best of our knowledge simple dipeptides without non- Fig. 1a), a water-soluble derivative of the common model com-
natural synthetic modifications are not known as minimalistic pound 2,4-dinitrophenyl acetate (DNPA), was chosen as the
esterase mimics. Based on our recent findings towards the out- model reaction. A solution of ANBS was added on top of the
standing self-aggregation properties of DKPs, we combined preformed hydrogel and reaction kinetics were monitored by
His-DKP 1 and Cys-DKPs (2, 3 and 4) as shown in Fig. 1b. UV/Vis. Initial Job’s plot analysis revealed a maximum initial
These three different blends [1 + 2], [1 + 3] and [1 + 4] should rate constant v0 at X = 0.4 for blend [1 + 2] and X = 0.5 for
give co-assembled nanostructures with a putative esterase blends [1 + 3] and [1 + 4] (Fig. 3A). We then investigated the
activity.21 We first investigated the principle co-aggregation pH-dependency of the ester hydrolysis (Fig. 3B). While with
properties of all three blends. Co-assembly was verified [1 + 2] v0 reaches a maximum at pH = 7.50, co-assemblies of
through hydrogel formation and subsequent investigation of [1 + 3] and [1 + 4] reached explicit maxima at slightly lower
the freeze-dried hydrogels via SEM (Fig. 2). All three blends pH-values (7.25 and 7.38).
formed stable hydrogels through a simple heating/cooling
In sharp contrast, v0 of pure self-assembled 1 has a
maximum at a pH of 6.50 which corresponds well to the pKa-
value of His. For self-assembled DKP 2 v0 increases until pH
7.5 and reaches a plateau. The broad maximum of Job’s plot
analysis for blend [1 + 2] together with the slight shift from
the theoretical optimal ratio of both DKPs from 1 : 1 as
observed for [1 + 3] and [1 + 4] is indicative for a random distri-
bution of 1 and 2 within the fibrous network (Fig. 4A). The
sharp maxima at X = 0.5 for [1 + 3] and [1 + 4] on the other
hand indicate a highly defined co-assembly of both DKPs
(Fig. 4B).
This defined alternating co-assembly should also result in a
better catalytic performance of blends [1 + 3] and [1 + 4], as
already indicated by the significantly higher v0-values. Next, we
wanted to compare v0 of the DKPs between the co-assembled
Fig. 1 (a) DKP-based mimic of a catalytic dyade (b) chemical structure
of investigated DKPs.
Fig. 2 SEM- and TEM-images of co-assembled DKP-blends A: [1 + 2];
B: [1 + 3]; C: [1 + 4].
Fig. 3 A: Job’s plot analysis of DKP-blends [1 + 2], [1 + 3] and [1 + 4]. B:
pH-dependency of the initial rate constants.
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exhibits a fast initial reaction turnover in the first 10 minutes
and finally approaches asymptotically a substrate conversion
that matches the total DKP concentration. Hence, only one
turnover is observed. With the same absolute molarity, the
corresponding hydrogel of 1 shows an inferior substrate con-
version, also with a strongly decelerating slope finally conver-
ging to an overall conversion close to the DKP concentration
(Fig. 5A, solid line). This diminished reactivity strongly indi-
cates the lower accessibility of the His-residues in the aggre-
gated state. The observed saturation in both the solution and
the gel state of 1 indicates a quick N-acetylation of the His-
residue followed by a very slow deacetylation, excluding cata-
lytic behaviour.
Next we investigated the corresponding blends (Fig. 5B–D).
In all cases the self-assembled blended DKPs were compared
with the corresponding DKPs kept in solution as a control. As
already observed for pure 1, all blended solutions, even though
accelerating ester hydrolysis, provided only one turnover.
Catalytic behaviour is only observed for blended hydrogels. For
[1 + 2] total conversion of the solution again converges to the
initial DKP-concentration while in the co-assembled state
product concentration exceeds DKP-concentration after 35 min
(Fig. 5B).
A similar catalytic behaviour was observed for [1 + 3] and
[1 + 4], although, as already indicated in Fig. 3A, ANBS hydro-
lysis was throughout faster, exceeding the initial DKP concen-
tration after 20–25 min. In sharp contrast to pure 1 and [1 + 2],
initial hydrolysis rates using the co-assembled blends [1 + 3]
and [1 + 4] were comparable to the solution phase experiments
(Fig. 5C and D). Overall, the co-assembled blend [1 + 4] shows
the best results in direct comparison with the corresponding
solution phase and in direct comparison to the other blends.
For a more precise comparison of their catalytic efficiency,
v0 was investigated in dependence of the substrate concen-
tration at the optimal pH and ratio for each blend. The
Michaelis–Menten enzyme kinetics model was used to calcu-
late the rate constants for all co-assembled hydrogels. In all
blends, catalyst turnover became the rate-limiting step at very
high substrate concentrations, a typical behaviour for enzyme-
catalysed reactions (see ESI – Table S2†). Michaelis Menten
constants (KM), rate constants (Kcat) as well as the catalytic
efficiencies (Kcat/KM) are given in Table 1.
Fig. 4 A: Random distribution of DKPs 1 and 2 within the co-assembly.
B: Alternating distribution of DKPs 1 and 3 or 4 within the co-assembly.
hydrogel state and a solution by disturbing the co-assembly
process through DMF addition. In general, v0 should be
reduced for the hydrogels since substrate availability is initially
strongly limited by diffusion processes. In addition, the acces-
sibility of the catalytically active His and Cys residues should
be strongly limited in the self-assembled hydrogel state
through intermolecular interactions of individual strands to
form the three-dimensional network. As an initial control
experiment, we tested the catalytic activity of pure His-DKP 1
in solution (Fig. 5A, dotted line). Even though 1 accelerated
ANBS hydrolysis, it cannot be defined as catalyst. The solution
The highest substrate-affinity and the highest catalytic
efficiency was once again observed for blend [1 + 4] (KM = 6.81).
Kcat values between [1 + 3] and [1 + 4] differ only insignifi-
cantly but KM is twofold higher for [1 + 3]. This is indicative for
Fig. 5 Product formation in ANBS hydrolysis, c (ANBS) = 60 mM; solid
lines: reaction was performed in the self-assembled hydrogel (gel);
dashed lines: reaction was performed in solution (sol) (HEPES : DMF =
1 : 1, V = 1.25 ml); dotted horizontal lines: total DKP concentration refer-
enced to the total volume; A: 1, pH = 6.50, c (1-hydrogel) = 92 mM; B: [1
+ 2] (1.5 : 1), pH = 7.50, c = 92 mM; C: [1 + 3] (1 : 1), pH = 7.25, c =
106 mM; D: [1 + 4] (1 : 1), pH = 7.38, c = 80 mM. Product conversion was
detected via UV/Vis at λ = 406 nm. In all experiments background
hydrolysis of ANBS was measured in the corresponding buffers at the
same pH with identical substrate concentration and subtracted.
Table 1 Summary of Michaelis–Menten kinetics
Hydrogel
KM (10−3 M)
Kcat (10−3 s−1
)
Kcat/KM (10−1 M−1 s−1
)
[1 + 2]a
[1 + 3]b
[1 + 4]c
8.51
12.18
6.81
0.73
1.60
1.46
0.86
1.31
2.14
a 1.5 : 1 ratio of 1 and 2, pH = 7.50. b 1 : 1 ratio of 1 and 3, pH = 7.25.
c 1 : 1 ratio of 1 and 4, pH = 7.38.
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a significantly weaker substrate affinity and might be the result
of sterically more favourable or multiple C–H-π-interactions
between 1 and 4 which subsequently leads to a closer proxi-
mity of the imidazole and thiol functionalities at the catalytic
active site of the dipeptide.
To verify this hypothesis, gas phase calculations based on
the low cost Hartree–Fock/minimal basis set composite
method HF-3C which shows excellent performance for nonco-
valent interactions have been accomplished for dimers of
[1 + 2], [1 + 3] and [1 + 4] (Fig. 6).22 Each energy minimized
structure confirms two central intermolecular H-bonds
between the two cyclic amides with typical O–H-distances
ranging from 1.74 to 1.92 Å. H-Bonds between the lipophilic
amino acids are significantly longer (1.89–1.92 Å) than the
H-bonds between the His and Cys amino acids (1.74–1.76 Å).
As predicted, all lipophilic side chains show significant C–H-
π-interactions. For [1 + 2] two C–H-π-interactions of the ortho-
and meta protons of the Phe side chain in 2 and the π-system
of the Phe side chain in 1 give a disordered T-shape geometry
between the two benzene rings with C–H-π-distances of 2.80
and 3.17 Å. In the calculated structure of [1 + 3] two significant
C–H-π-interaction between two C–Hγ protons of the terminal
CH3-group of the Val side chain in 3 and the benzene ring in 1
exist. The calculated C–H-π-distances to the centroid of the
benzene ring is 3.04 Å and 3.05 Å to the centroid of the C3–C4-
Fig. 7 A: Detail magnifications of 1H HRMAS NOE spectra in D2O of A:
Hydrogel [1 + 3] (1 : 1) and B: Hydrogel [1 + 4] (1 : 1); diamonds: aromatic
protons of the Phe sidechain of 1, triangles: C–Hγ protons of the Val
sidechain of 3, circles: C–Hδ protons of the Leu sidechain of 4.
π-bond. For [1 + 4], two C–H-π-interaction are observed with C–
H-π-distances of 2.70 and 2.77 Å between C–Hδ protons of both
terminal CH3 groups and two distinct C–C-π-bonds of Phe. All
distances are in good agreement with typical average distances
of C–H-π interactions as observed in solid state protein struc-
tures.23 Obviously, the additional methylene group in the side
chain of 4 allows a significantly stronger C–H-π-interaction as
implicated by shorter C–H-centroid distances.
In all blends, combination of the two central amide hydro-
gen bonds and the additional C–H-π-interaction arranges the
functional imidazole and thiol functionalities into close proxi-
mity. Calculated S–H–N-distances vary from 2.18 Å in [1 + 2]
and [1 + 3], and 2.07 Å for [1 + 4]. It has to be mentioned, that
the horizontal dimension of these calculated single-strands
(approx. 1 nm) is one dimension below the observed fibre
thickness as observed via SEM and TEM. Thus, further inter-
strand interactions must be operational which strongly limits
the true accessibility of the catalytically active sides in the self-
assembled state. Under this premise it is even more surprising
that blends [1 + 3] and [1 + 4] show similar initial hydrolysis
rates in comparison to the corresponding DKPs kept in
solution.
To finally verify that the calculated alternating co-assembly
in [1 + 3] (Fig. 7A) and [1 + 4] (Fig. 7B) is based on C–H-
π-interactions, 1H HRMAS NOESY experiments of the co-
assembled hydrogels were performed in D2O. Clearly, the
strongest NOE correlation was observed between C–Hγ of 3 or
C–Hδ of 4 and C–Haryl of 1 supporting the calculated structure
of this blend.24
Conclusions
In summary we described the most minimalistic peptide self-
assembly with an enzyme-like activity. It is based on abiogen-
esis relevant cyclic dipeptides solely build from the proteino-
genic amino acids Phe, His, Val, Leu and Cys. A catalytic turn-
over is exclusively observed in the self-aggregated state for het-
erologous mixtures (blends) of His- and a Cys-containing cyclic
dipeptides. Hartree–Fock calculations as well as HRMAS NOE
experiments strongly indicate that C–H-π-interactions as well
Fig. 6 Calculated structures of DKP-dimers. A: [1 + 2]; B: [1 + 3]; C: [1 +
4]. Structures were calculated using the semi-empirical HF-3c functional
in the gas phase.
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as intermolecular amide hydrogen bonds are responsible for
the heterologous self-aggregation which finally leads to a close
proximity of His and Cys side chain to give a catalytic dyade.
These findings offer a new understanding toward a potential
role of the so far undervalued role of cyclic dipeptides in
chemical evolution and further implies the importance of self-
assembled peptide aggregates in the pre-Darwin evolution.
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Financial Support by the Fonds der Chemischen Industrie
(FCI) is gratefully acknowledged.
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