Protein self-assembly induces promiscuous nucleophilic biocatalysis in Morita-Baylis-Hillman (MBH) reaction

Article abstract of DOI:10.1039/c5ra23949d

Self-assembled states of proteins render efficient promiscuous nucleophilic biocatalysis in MBH reaction in a green process. The His and Arg based catalophores in proteins operate in aqueous buffer at neutral pH and ambient temperature. Steady-state fluor

Full text of DOI:10.1039/c5ra23949d

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Protein self-assembly induces promiscuous
nucleophilic biocatalysis in Morita–Baylis–Hillman
(MBH) reaction†
Cite this: RSC Adv., 2016, 6, 208
Received 13th November 2015
Accepted 11th December 2015
Pralhad N. Joshi, Landa Purushottam, Nirmal K. Das, Saptarshi Mukherjee
*
and Vishal Rai
DOI: 10.1039/c5ra23949d
www.rsc.org/advances
Self-assembled states of proteins render efficient promiscuous nucleophilic residues where histidine (His) serves as a potential
nucleophilic biocatalysis in MBH reaction in a green process. The His candidate for nucleophilic catalysis. However, the earlier efforts
and Arg based catalophores in proteins operate in aqueous buffer at (Fig. 1a) with His or His tag bearing proteins and our initial
neutral pH and ambient temperature. Steady-state fluorimetric investigations (Table S1, ESI†) suggested that His alone is not
approaches reveal that lower order aggregates play a seminal role in suitable for catalyzing the MBH reaction.
the biocatalytic process.
Here, we report promiscuous catalysis by lysozyme C 5c and
myoglobin 5e hinged on catalophore formed by His and Argi-
nine (Arg) pair to catalyze the MBH reaction with remarkably
high efficiency. A set of control experiments (Table S1, ESI†)
suggested that Arg can potentially mediate proton shuttling
process in the transition state10 (4) and assist His for the
necessary activation (Fig. 1b). The uorimetric investigations
indicate that the lower order assembly of catalophore consisting
protein serves as an active catalyst. These results invite recon-
sideration of the catalytic species in the eld of biocatalysis and
pave the path for its potential integration with protein
aggregation.
Initially, we selected a variety of proteins (5a–5e, Table 1) that
offer His and Arg pair conned variably in the conformational
space. Cyclohexenone 2 and p-nitrobenzaldehyde 1a were
selected as Michael acceptor and electrophile, respectively.
When RNase A 5a was employed as a catalyst, 35% conversion to
MBH adduct was observed (entry 1, Table 1). His and Arg resi-
The chemical industry has seen a remarkable increase in the
use of biocatalysis in recent years.1 Structurally dened spatial
distribution of residues in proteins has offered efficient catal-
ysis for a variety of mechanistically unique synthetic trans-
formations.2–4 Over a period, the perspective of high specicity
in enzyme catalysis has changed and led to the realization that
a large population of enzymes is capable of catalyzing multiple
reactions.5 However, non-biological transformations have
posed a stiff challenge to biocatalysis. Promiscuous enzymes or
protein-based catalysts are key aspirants in this case and have
drawn attention from diverse streams of Science.6 One of the
most important synthetic transformations, carbon–carbon
bond formation, is realized in natural systems7 but do not
extend to non-biological chemical transformations. For
example, MBH reaction has been a notoriously difficult target
owing to a sequence of steps that involves mechanism switching
based on the substrates and solvents. MBH reaction involves
nucleophilic catalysis in a Michael addition rendering enolate
that entraps an electrophile to form C–C bond in an aldol
condensation and results in a functionally rich molecule. There
are no examples of antibodies that can catalyze this reaction. A
recent attempt in this direction through directed evolution of
engineered enone-binding proteins8 and another previous
attempt9 met limited success. Protein backbone has several
˚
dues in RNase A (PDB ID : 2AAS) are separated by ꢀ11.3 A in
a constrained microenvironment, with a lysine (Lys) side chain
Department of Chemistry, Indian Institute of Science Education and Research (IISER)
Bhopal, Bhopal Bypass Road, Bhauri, Bhopal, MP 462 066, India. E-mail: vrai@iiserb.
ac.in
Fig. 1 (a) Biocatalysis in MBH reaction. (b) Hypothetical activation of
nucleophile and electrophile in a Michael addition–aldol condensation
sequence (MBH) draws comparison with the catalytic activity of
lipases.11
† Electronic supplementary information (ESI) available: Methods, procedures,
additional results and discussion, characterization data. See DOI:
10.1039/c5ra23949d
208 | RSC Adv., 2016, 6, 208–211
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Table Identification of protein that renders His and Arg pair
RSC Advances
1
conversions (20%) comparable to 5f and 5g, thereby substanti-
ating the role of His in catalysis. It was evident from these
results that both the presence and inter-residual distance/
orientation between His and Arg are the driving parameters in
controlling the efficiency of the catalytic process.
appropriate for MBH reaction
The concentration of the reaction mixture was optimized
with respect to the protein (Table 2). The optimal concentration
% conversion was found to be 2.5 mM for lysozyme C 5c and 1 mM for
His and
Arg pair
Entry Catalysta
(3a)b
myoglobin 5e (entries 5 and 4). We observed striking non-linear
change in efficiency of catalyst from 1 to 2.5 mM for lysozyme
C 5c and 500 mM to 1 mM in case of myoglobin 5e. The results
1
2
3
4
5
6
7
RNase A 5a
a-Chymotrypsinogen 5b 1 (H40, R145)
Lysozyme C 5c
a-Chymotrypsin 5d
Myoglobin 5e
Trypsin 5f
1 (H119, R10)
35
53
72
indicated that protein assembly rendered a constitution of the
active catalyst.13 The aggregation phenomenon of proteins and
peptides has been investigated with techniques such as ultra-
centrifugation,14 dialysis,15 Gouy interferometry,16 neutron
scattering,17 NMR dispersion,18 NMR diffusion19 and uorim-
etry.20 A clear mechanistic understanding in this area is still
elusive, but there is a consensus about aggregation with respect
to the concentration. It outlines that 1.0 mM lysozyme C 5c
solution can be considered unsaturated primarily having
monomers whereas, 2.5 mM solution can be regarded as satu-
rated having lysozyme C 5c assemblies.
1 (H15, R14)
2 (H91, R93; H65, R217) 37 ꢁ 10
2 (H24, R118; H113, R31) 65
0
0
16 ꢁ 8
Ubiquitin 5g
22
a
Protein concentration (2.5 mM). b % conversion by 1H NMR. Relative
stoichiometry of 1a and 2 is 1 : 3 aer optimization. 1a and 2 were
dissolved in DMSO (buffer : DMSO, 9 : 1) for all the reported reactions
in this paper (see ESI for details). For catalyst loading, see Tables S2
and S3 in ESI.
We believe that lysozyme C 5c assemblage leads to the
observed catalytic activity and that too at a specic concentra-
tion of 5c, as observed herein. In order to have a better mech-
anistic understanding of the actual process of aggregation the
protein undergoes, we have carried out thioavin T (ThT) assays
at various concentrations of lysozyme C 5c (0.5–5 mM). The ThT
uorescence spectra in the presence of varying concentrations
of myoglobin 5e were too structured having multiple well-
dened peaks. Hence, to avoid the over-interpretation of data
owing to the aggregation process, we did not carry out the
uorescence studies with myoglobin further. The uorescent
probe, ThT is unique in the sense that it has been widely used to
unravel the nuances associated with the process of self-
assembly and/or aggregation of proteins and peptides.20 The
dramatic increment of uorescence intensity of ThT upon
Fig. 2 His (blue) and Arg (red) pair(s) in lysozyme C 5c and myoglobin
5e. (See Fig. S1† for 5a, 5b, 5d and 5f–g).
placed between them. a-Chymotrypsinogen 5b (PDB ID : 1EX3)
˚
aligns these residues at ꢀ8.3 A with no interfering residue and
resulted in 18% improvement in the conversion to MBH adduct
(entry 2). Lysozyme C 5c (PDB ID : 2ZYP, Fig. 2), where the
Table 2 Effect of protein concentration on MBH reaction
˚
two residues are separated only by ꢀ4.2 A resulted in remark-
able improvement with 72% conversion (entry 3, Table 1). The
inter-residue distance is only of qualitative interest as solvent
accessibility or dynamics of the domain is not taken into
account. Although a-chymotrypsin 5d (PDB ID : 4CHA) and
myoglobin 5e (PDB ID : 1MBO) have two sets of His and Arg
pairs, yet in the former, the residues were reasonably separated
as compared to the latter. Perhaps, this explains why myoglobin
5e (Fig. 2) turned out to be a better catalyst than a-chymotrypsin
5d (entries 4 and 5, Table 1). Besides, we realized that both
a-chymotrypsin 5d and trypsin 5f result in capricious conver-
sions in multiple attempts due to self-digestion of proteins.
In a control experiment, we selected proteins in which none
of the His and Arg residues are in close vicinity (entries 6 and 7).
In both the cases, poor reactivity was observed. In yet another
control experiment, we blocked the nucleophilic site of
His15 in lysozyme C 5c by a chemoselective transformation
% conversion (3a)b,c
Entry
Concentrationa
Lysozyme C (5c)
Myoglobin (5e)
1
2
3
4
5
6
7
50 mM
100 mM
500 mM
1 mM
2.5 mM
5 mM
0
0
8
10
72
27
25
0
0
9
79
65
55
30
10 mM
Concentration with respect to the protein. b % conversion determined
a
by 1H NMR. No background reaction was observed in catalyst free
c
with 2,40-dibromoacetophenone.12 The modied 5c resulted in condition at all the concentrations.
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binding to the b-rich deposits makes it an excellent tool for incubation) that the uorescence intensities of Trp decreases
deciphering self-aggregation phenomenon. It is believed that upon increasing the protein concentration (Fig. S4, ESI†).
ThT not only binds to a variety of substrates but also the Interestingly, here also, we did not observe any appreciable
micellar form (its critical micelle concentration is ꢀ4 mM in change in the emission maxima of Trp indicating that even
water) interacts with the brils.20a Lysozyme C 5c in its native upon aggregation, the uorophore does not either undergo
state is composed of ꢀ32% a-helix and ꢀ7% b-sheet21 and major internalization or gets exposed to the hydrophilic envi-
due to aggregation, the content of the latter increases dramat- ronment. The gradual fall in the uorescence intensities of
ically which gets reected by the rise in uorescence intensity of Trp with a rise in lysozyme C 5c concentration can be attributed
ThT (Fig. S2, ESI†).
to the self-quenching brought in by the additional amino acid
Fig. 3a represents the variation of uorescence intensities residues that are in close proximity of the uorophore. To have
against time for different concentrations of lysozyme C 5c used a molecular level interpretation of our steady-state spectro-
in the present investigation. As seen from the gure, the uo- scopic data, we carried out molecular docking studies
rescence intensity of ThT at any instant rises almost linearly (Fig. 3b).23 ThT binds at a distance of 21.9 A and 24.8 A from His
with increasing concentration of the protein that is a clear and Arg residues of catalophore, respectively and the estimated
signature of the concentration-driven oligomerization that the free energy of binding is ꢂ7.91 kcal molꢂ1. Although ThT is
protein undergoes. The oligomeric states are largely stable over located substantially away from the catalytic site, nonetheless, it
a period of 72 hours. It must be stated here that the emission is capable of reporting the increased concentration-induced
maxima of ThT (Fig. S2, ESI†) is not a function of lysozyme C 5c aggregation as evidenced from the spectroscopic data. The
concentration, thereby signifying that the microenvironment microenvironment of both ThT and Trp (both being in close
of the uorophore remains almost unaltered due to aggrega- vicinity to each other) does not change due to aggregation.
tion. The process of aggregation goes beyond the catalytic Thus, a linear assembly along the X–Y plane encompassing the
maxima of 2.5 mM lysozyme C 5c and the continued rise in catalophore can be envisaged. The increase in lysozyme C 5c
uorescence intensities of ThT beyond that point is a signature concentration induces higher order aggregation, however, the
of the increased b-sheet content as a result of aggregation. catalytic site gets deactivated likely due to excessive crowding
Lysozyme C 5c, an intrinsically uorescent protein has six and hence the catalytic efficiency goes through a maxima
tryptophan (Trp) residues. Out of these, only two Trp62 and (at ꢀ2.5 mM lysozyme C 5c, entry 5, Table 2). The diameter of 5c
Trp108 are dominant and located in the substrate binding assembly at 2.5 mM was determined to be 10.7 nm (Fig. S6†) by
site.22 We also monitored the emission characteristics of Trp as dynamic light scattering (DLS) experiments. Electron micros-
a function of increasing concentration of lysozyme C 5c (Fig. S3, copy (FE-SEM) was not found suitable to probe these systems
ESI†) and observed (spectra were recorded till 72 hours of due to enhancement in order of aggregation during sample
preparation (Fig. S5†).
˚
˚
The data suggest that the lower order assemblies play a crucial
role in catalysis. This is likely enabled by the forged hydrophobic
cavities in these protein constructs (lysozyme C 5c at 2.5 mM) that
allow increased effective concentration of reagents. A sharp
decrease in efficiency with higher order assembly of 5c (5 mM)
reaffirms that the domain with catalophore is involved in
aggregation as complemented by the uorimetric analysis. A
subtle balance of protein concentration is apparently crucial for
the success of these catalytic transformations.
With the concentration of protein as a xed parameter, we
varied the amount of catalyst and found 10 mol% of lysozyme
C 5c and 2–10 mol% of myoglobin 5e were efficient (Table S2,
ESI†). The optimized conditions were then examined for their
application to a variety of substrates (Scheme 1). It was
intriguing to see that lysozyme C 5c and myoglobin 5e were able
to catalyze MBH reaction with a broad range of electronically
diverse aldehydes. The relative catalytic efficiency of lysozyme C
5c and myoglobin 5e is dependent on the choice of aldehydes.
The latter was superior in multiple cases (3a, 3c–e, 3g–h, 3j–n
and 3p) whereas at other instances, both 5c and 5e were equally
efficient. Interestingly, 2 mol% of myoglobin was more efficient
than 10 mol% in two cases (3b, 3d) indicating towards the
substrate dependent behaviour of protein assemblies.
Fig. 3 (a) ThT fluorescence assay with increasing concentration of
lysozyme C 5c as a function of time. The colour black, red, blue, cyan,
pink, yellow, and navy blue represents the concentration of lysozyme
It was evident that the reactivity prole offered by these two
proteins was unique with respect to other nucleophilic catalysts
used in MBH reaction. To substantiate this point, we selected
C 0.5, 1, 1.5, 2, 2.5, 3, 5 mM, respectively. (b) Energy minimized
molecular docked structure of lysozyme C with ThT.
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Acknowledgements
P. N. J., L. P. and N. K. D. are recipient of research fellowship
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