A Hydroxyquinoline-Based Unnatural Amino Acid for the Design of Novel Artificial Metalloenzymes

Article abstract of DOI:10.1002/cbic.202000306

We have examined the potential of the noncanonical amino acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial metalloenzymes. HQAla, a versatile chelator of late transition metals, was introduced into the lactococcal multidrug-resista

Full text of DOI:10.1002/cbic.202000306

Combining Chemistry and Biology
Accepted Article
Title: A hydroxyquinoline-based unnatural amino acid for the design of
novel artificial metalloenzymes
Authors: Ivana Drienovská, Remkes A. Scheele, Cora Gutiérrez de
Souza, and Gerard Roelfes
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemBioChem 10.1002/cbic.202000306
Link to VoR: https://doi.org/10.1002/cbic.202000306
01/2020

10.1002/cbic.202000306
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COMMUNICATION
A hydroxyquinoline-based unnatural amino acid for the design of
novel artificial metalloenzymes
Ivana Drienovská[a],†, Remkes A. Scheele[a],†, Cora Gutiérrez de Souza[a] and Gerard Roelfes*[a]
[a]
Prof. G. Roelfes, Dr. I. Drienovská, R. A. Scheele, C. Gutiérrez de Souza
Stratingh Institute for Chemistry,
University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
E-mail: j.g.roelfes@rug.nl
[†]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Abstract: Here we examine the potential of the non-canonical amino
acid (8-hydroxyquinolin-3-yl)alanine (HQAla) for the design of artificial
metalloenzymes. HQAla, a versatile chelator of late transition metals,
was introduced into the lactococcal multidrug resistance regulator
(LmrR) via stop codon suppression methodology. LmrR_HQAla was
shown to efficiently complex with three different metal ions, CuII, ZnII
and RhIII to form unique artificial metalloenzymes. The catalytic
potential of the CuII bound LmrR_HQAla enzyme was shown through
its ability to catalyze asymmetric Friedel-Craft alkylation and water
addition reactions, whereas the ZnII coupled enzyme was shown to
mimic natural Zn-hydrolase activity.
Z-domain protein. Here it was used as a fluorescent probe and to
bind heavy metals for crystallography.[10] The constitutional
isomer of this ncAA, 2-amino-3-(8-hydroxyquinolin-5-yl)propanoic
acid, has been described and used in protein electron transfer and
ZnII sensing in vitro and in vivo.[11]
Metalloproteins represent more than one-third of natural enzymes,
catalysing many of the important chemical reactions that sustain
life.[1] This has inspired the creation of artificial metallo-enzymes -
hybrids of proteins and synthetic transition metal complexes,
ultimately aiming to develop biocatalysts for reactions that are
new-to-nature.[2]
Synthetic transition metal complexes have been
incorporated into protein scaffolds using different methods to bind
the metal-ligand complexes.[3] A recent approach to introduce
metal catalysts involves non-canonical amino acids (ncAAs),
either serving as the ligand which directly chelates the metal, or
by binding an external metal-binding ligand. The ncAAs are
introduced using expanded genetic code methods, especially
stop codon suppression.[4]
Figure 1. Schematic representation of the proposed design for novel artificial
metalloenzymes. Surface representation of LmrR protein scaffold (PDB: 3F8B)
with positions chosen for incorporation of HQAla highlighted in orange (V15)
and magenta (M89).
Several metal-binding ncAAs have been incorporated in
proteins in vivo via expanded genetic code methodology to create
artificial metalloenzymes.[5] For example, the amino acid
analogue of the bipyridine ligand, (2,2΄-bipyridin-5-yl)alanine
(BpyA), has been incorporated into different biomolecular
scaffolds to bind different bivalent metalsand catalyse a variety of
reactions when complexed with CuII.[6] Likewise, the incorporation
So far, expansion of the genetic code with HQAla has not
resulted in designer biocatalysts. Here, we introduce HQAla into
Lactococcal multidrug resistance regulator (LmrR)[12] and
complex it with a variety of transition metal ions such as CuII, ZnII
and RhIII. The catalytic propensity of the CuII bound enzymes was
demonstrated in a vinylogous Friedel-Crafts alkylation and an
enantioselective hydration reaction. The ZnII bound artificial
metalloenzyme was shown to hydrolyse amide bonds.
The artificial metalloenzymes presented in this study were
prepared utilizing amber stop codon methodology, using a
plasmid carrying the orthogonal tRNA-synthetase/tRNA pair
specific for the incorporation of HQAla (pEVOL-HQAla).[10] HQAla
was synthesized according to a previously reported method with
a few minor improvements.[10] The positions M89 and V15 of LmrR
were selected for the incorporation of HQAla, as these positions
have been previously shown to allow efficient incorporation of
ncAAs.[6ef,13] Both residues are pointing towards the inside of the
hydrophobic pore of the protein, with M89 located at the far edge
and V15 more to the middle of the pore. The M89 position was
shown to be the optimal position for studies with BpyA,[6ef] while
of
non-canonical
N-methylhistidine
and
3,4-
dihydroxyphenylalanine into metallo-enzymatic scaffolds has
been shown to both boost enzymatic turnover, and facilitate
reutilisation of an alcohol dehydrogenase active site to bind ZnII
respectively.[7]
The metal ligand 8-hydroxyquinoline (HQ) is one of the
earliest analytical reagents and has been shown to bind >20
transition metals.[8] More recently, the strong N-O bidentate
binding mode of HQAla has been utilized for a variety of CuII, ZnII
and Rh mediated reactions, highlighting the versatility of HQ.[9]
The ncAA that employs 8-hydroxyquinoline as its functional group,
2-amino-3-(8-hydroxyquinolin-3-yl)propanoic
acid
(HQAla)
(Figure 1) was first incorporated as unnatural amino acid into the
1
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position V15 has been recently described as the optimal position
for p-aminophenylalanine incorporation.[13]
The pEVOL-HQAla plasmid and
a pET17b plasmid
containing either LmrR_V15TAG or LmrR_M89TAG constructs
were contransformed into E. coli BL21 C43(DE3). The LmrR
variant used in this study contains two mutations, K55D and K59Q,
which are introduced to remove the DNA binding ability of LmrR,
and a C-terminal Strep-Tag.[14] After addition of HQAla to the
media, the cells were induced to produce LmrR_V15HQAla or
LmrR_M89HQAla (Figure S1). The expression yields were 18-22
mg/L for LmrR_V15HQAla and 4-10 mg/L for LmrR_M89HQAla.
The lower yield of LmrR_M89HQAla is mainly attributed to faulty
translation, i.e failure to suppress the stop codon TAG, resulting
in truncated LmrR(1-88). The incorporation of HQAla was
confirmed by electrospray ionization mass spectrometry (ESI-MS)
(Figure S2). The quaternary structure of LmrR was studied by
analytical size exclusion chromatography to determine the effect
of unnatural amino acid incorporation. It was found that the
structure was preserved and the proteins were eluted as single
peaks at 11.4 (± 0.1) ml, which represents a molecular weight of
approximately 30 kDa, consistent with dimeric LmrR (Figure S3).
The ability of LmrR_V15HQAla and LmrR_M89HQAla to
bind metal salts was studied by UV-Vis titrations, measuring the
change in the UV-Vis absorption spectrum upon addition of the
metal salts. The titrations were performed with Cu(NO3)2,
Zn(NO3)2, Cp*RhCl2, RhCl(COD), Rh2(AcO)4 and Rh2Cl2(CO)4.
Titration of Cu(NO3)2 to LmrR_V15HQAla or LmrR_M89HQAla
caused hypochromic shifts to the ligand-centered (LC) charge
transfer band at 249 and to a lesser extent the LC band at 320,
arising from π-π* and n-π* transitions respectively.
Simultaniously, hyperchromic shifts of ligand-to-metal charge
transfer (LMCT) transitions at 269 nm and to a lesser extent at
~390 nm were observed. (Figure 2ab, Figure S4).[15] These shifts
are in agreement with complex formation of metals to the
quinoline moiety involving deprotonation of the phenolic group.[16]
When approximately one equivalent of Cu(NO3)2 with respect to
the protein monomer was added, no more changes were
observed in the spectrum. Furthermore, LmrR without HQAla
showed no changes in absorption upon addition of Cu(NO3)2
(Figure 2c). This suggests that the HQAla residue is the preferred
CuII binding site, albeit that some unspecific binding to other parts
of the protein, which is not detectable by UV-Vis measurements,
cannot be excluded. Using the same method, Zn(NO3)2 and the
rhodium complexes were titrated against LmrR_V15/M89HQAla
and LmrR (Figures S4 and S5). Complexing of HQala with
Zn(NO3)2 and Cp*RhCl2 caused similar hypo/hyper-cromic shifts
until one equivalent of metal was added, indicating binding
accomponied by deprotonation. Again, no changes in absorption
were observed when titrating either metal against LmrR,
indicating binding of ZnII and rhodium-bound cyclopentadienyl to
the HQ moiety in LmrR specifically. Rh2(AcO)4 and Rh2Cl2(CO)4
did not seem to bind LmrR or HQAla, since the absorption spectra
of both LmrR_V15HQAla and LmrR did not change upon titration
with these rhodium salts. RhCl(COD) behaved different, altering
the absorbance of LmrR_V15HQAla and LmrR beyond the
addition of 1 equivalent, and was therefore thought to bind
unspecifically to the scaffold. Taken together, the versality of
HQAla to bind metal ligands proved to be effective in capturing
CuII, ZnII or Cp*RhIII into the dimeric LmrR scaffold.
Figure 2. UV-visible titrations with Cu(NO3)2 with a) LmrR_V15HQAla, b)
LmrR_M89HQAla and c) LmrR. Insets show the plots of the absorbance at 269
nm as function of the equivalents of Cu(NO3)2 added.
The catalytic potential of these novel artificial
metalloenzymes was studied in several reactions. Previous
functionalisation of LmrR with a covalently attached Rh catalyst
[17]
was successful in the catalytic hydrogenation of CO2,
whereas
the water stable Cp*Rh complex was used to accelerate aromatic
C-H activation when bound by streptavidin.[18] However, testing
LmrR_V15/M89HQAla complexed with Cp*Rh for the same,
aromatic C-H activation did not gie rise to detectable product
formation, most likely due to the limited number of free
coordination sites when Cp*Rh is bound to HQAla.
ZnII serves as the catalytic metal in numerous different
hydrolases.[19] Therefore, we decided to probe the hydrolytic
potential of LmrR_HQala_ZnII on five different substrates. First,
the hydrolysis of ester bonds was studied, using two model
substrates: p-nitrophenyl acetate and p-nitrophenyl butyrate.
Although LmrR_HQala_ZnII hydrolyses these substrates with
rates 3-3.5x higher than the uncatalyzed reaction, the fact that
unbound Zn(NO3)2 with LmrR yielded similar rate accelarations
indicated that the specific active site was not required for these
reactions to proceed. The hydrolysis of p-nitrophenylphosphate
was tested, however no hydrolysis was observed, with or without
the metal bound.
2
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10.1002/cbic.202000306
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(Table 1, entry 3). LmrR_M89HQAla_Cu(II) gave rise to 25% ee
and 20% conversion (Table 1, entry 4). While the conversion is
lower than in the reaction catalyzed by Cu(NO3)2, the
enantioselectivity proves the benefit of the reaction occurring in a
chiral scaffold.
The water addition (Scheme 1b) was catalyzed by
Cu(NO3)2 to give 84% conversion (Table 1, entry 6). With CuII
bound to LmrR_V15HQAla, a small decrease in activity was
observed while the enantioselectivity stayed very low (Table 2,
entry
7).
Similar
to
the
Friedel-Crafts
alkylation,
LmrR_M89HQAla_CuII lowered the yield, but concomitantly
increased the ee (51%) (Table 2, entry 8).
Figure 3. Substrates used for the amide-bond hydrolysis reaction.
Next, the ability of LmrR_V15/M89HQAla_ZnII to hydrolyze
amide bonds was examined using substrates 1 and 2 (Figure 3).
Both of these substrates are small peptides with 2 or 3 extra
amino acids bound to p-nitrophenylalanine. Hydrolysis of these
Overall, our novel artificial copper enzymes were successful
in the catalysis of both studied reactions. Notably, similar trends
were observed between the V15 and M89 mutants. With the
LmrR_V15HQAla mutant, high conversions were obtained. These
results can be explained either by ideal localization of the central
tryptophanes contributing to substrate binding, or by the HQAla
moiety being more solvent exposed at this position. The latter
seems more likely because of the low enantioselectivity of the
reactions with this mutant. However, this hypothesis should
ideally be confirmed with X-ray structures. The low conversions
with LmrR_M89HQAla suggest that HQAla incorporated at this
position is less accessible, or does not form interactions
favourable for catalysis, however this more constrained position
does facilitate enantioselective product formation.
substrates
produces
p-nitroaniline
which
can
be
spectrophotometrically followed at 410 nm. Surprisingly, while the
hydrolysed product of 1 was not observed, we did observe (slow)
hydrolysis of substrate
2
(Figure S7). While the
LmrR_V15/M89HQAla_ZnII enzymes were active on substrate 2,
no p-nitroaniline was produced when LmrR with Zn(NO3)2 was
added. Therefore, the hydrolysis of 2 is thought to rely on the ZnII
containing active site in LmrR_HQala.
Finally, the catalytic activity of CuII/HQAla-containing
artificial metalloenzymes was evaluated in the vinylogous Friedel-
Crafts alkylation reaction of 5-methoxy-1H-indole (4) with 1-(1-
methyl-1H-imidazol-2-yl)but-2-en-1-one (3) (Scheme 1a),[6e,20]
and the 1,4-addition of water to α,β-unsaturated 2-acyl pyridine 6
resulting in the corresponding β-hydroxy ketone product 7
(Scheme 1b).[6f,14,21]
Table 1. Results of the vinylogous Friedel-Crafts reaction of 3 and 4 resulting in
5 and of the conjugate addition reaction of water to 6 resulting in 7, both
catalyzed by LmrR_V15HQAla and LmrR_M89HQAla.
Conv.
(%)
ee
(%)
Entry
Catalyst
Substrate
Product
1
2
3
4
5
6
7
8
-
3,4
3,4
3,4
3,4
6
5
5
5
5
7
7
7
7
4 ± 2
99 ± 1
97 ± 1
20 ± 2
11 ± 3
84 ± 7
77 ± 8
20 ± 5
-
Cu(NO3)2
-
LmrR_V15HQAla_CuII
LmrR_M89HQAla_CuII
-
<5
25 ± 3
-
Cu(NO3)2
6
-
LmrR_V15HQAla_CuII
LmrR_M89HQAla_CuII
6
<5
6
51 ± 10
[a] Typical conditions: 9 mol% Cu(H2O)6(NO3)2 (90 µM) loading with 1.25 eq
LmrR variant in 20 mM MOPS buffer, 150 mM NaCl, pH 7.0 for 1 day at 4 °C.
All data are the average of two independent experiments, each carried out in
duplicate, reporting the average conversion/ee with their respective standard
deviation.
Scheme 1. a) Artificial metalloenzyme catalyzed vinylogous Friedel-Crafts
reaction and, b) Water-addition reaction. Reactions were run in 3-(N-
morpholine)propanesulfonic acid (MOPS) buffer (20 mM, 150 mM NaCl, pH
7.5). After incubation of Cu(NO3)2 with the protein for 2 hours, the relevant
substrates were added and reactions were run at 4 °C for 24 hours.
In summary, we have presented novel artificial
metalloenzymes containing the non-canonical amino acid HQAla
as metal-binding moiety. The catalytic potential of these newly
created artificial metalloenzymes was evaluated in various
reactions. HQAla was successfully incorporated into the structure
of LmrR at two different positions: V15 and M89. Both mutants of
LmrR showed a good affinity for different metal salts, such as
Cu(NO3)2, Zn(NO3)2 and Cp*RhCl2. The novel ZnII-containing
artificial metalloenzymes showed activity in the hydrolysis of
peptide bonds while CuII-containing variants showed activity in
Friedel-Crafts alkylation reaction of 5-methoxyindole with α,β-
unsaturated-2-acyl imidazole and water-addition reaction to α,β-
unsaturated 2-acyl pyridine, although with low to moderate
enantioselectivities. Overall, due to the high affinity of
LmrR_HQAla towards the different metal salts and complexes,
Both reactions were carried out using 9 mol% of Cu(NO3)2
(90 µM) with
a
small excess of LmrR_V15HQAla or
LmrR_M89HQAla (112.5 µM of the monomer). The uncatalyzed
Friedel–Crafts alkylation proceeds with 4% conversion, wheras
the unbound Cu(NO3)2 catalyzed this reaction to give 99%
conversion (Table 1, entry 1-2). The reaction catalyzed by
LmrR_V15HQAla_CuII yielded similar results to those with
Cu(NO3)2 in solution, suggesting that although CuII binds to HQAla
in the hydrophobic pore (Figure 2a), the metal-catalyst is situated
too far from the chiral scaffold to influence chirality of the reaction.
3
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10.1002/cbic.202000306
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COMMUNICATION
this artificial metalloenzyme can provide a platform for a range of
different, currently unexplored, metal-catalyzed reactions. We
believe that HQAla, together with other known metal-binding
ncAAs, open up the way for a variety of (new-to-nature)
biotransformations.
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Acknowledgements
This work was supported by the Netherlands Organization for
Scientific Research (NWO, vici grant 724.013.003) and the
European Research Council (ERC starting grant 280010). GR
acknowledges support from the Netherlands Ministry of Education,
Culture and Science (Gravitation program no. 024.001.035). The
authors thank Prof. P. G. Schultz (The Scripps Research Institute,
USA) for kindly providing the pEVOL_HQAla plasmid and Ruben
Maaskant for useful suggestions and discussion regarding the
synthesis of HQAla.
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4
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
Insert text for Table of Contents here.
Several metal-binding non-canonical amino acids have been incorporated in protein scaffolds to create artificial metalloenzymes. Here,
we introduce 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQAla) into lactococcal multidrug resistance regulator (LmrR) and
complex it with several transition metal ions such as CuII, ZnII and RhIII to showcase its catalytic potential in a variety of different
reactions.
Institute and/or researcher Twitter usernames: @IvanaDrienovska @RoelfesGroup
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