Development of a simple high-throughput assay for directed evolution of enantioselective sulfoxide reductases

Article abstract of DOI:10.1039/d0cc01660h

We report on the development of high-throughput fluorogenic assay that can streamline directed evolution of enantioselective sulfoxide reductases. As a model, methionine sulfoxide reductase A (MsrA) has been evolved to expand its limited substrate scope. The resulting mutant MsrA can resolve a range of new challenging racemic sulfoxides with high efficiency including the pharmaceutically relevant albendazole sulfoxide. The simplicity and the level of throughput make this method also suitable for the screening of metagenomic libraries in future for the discovery of new enzymes with similar reactivities.

Full text of DOI:10.1039/d0cc01660h

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DOI: 10.1039/D0CC01660H
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Development of a Simple High-Throughput Assay for Directed
Evolution of Enantioselective Sulfoxide Reductases
Vincenzo Tarallo, Kasireddy Sudarshan, Vladimír Nosek and Jiří Míšek *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We report on the development of high-throughput fluorogenic wide range of substrates. The nature of the substituent at the
assay that can streamline directed evolution of enantioselective R1 site of the sulfoxide moiety does not significantly affect the
sulfoxide reductases. As a model, we have evolved methionine activity of the enzyme but the only tolerated substituents at the
sulfoxide reductase A (MsrA) to expand its limited substrate scope. R2 site are methyl and ethyl groups (Scheme 1). Sterically more
The resulting mutant MsrA can resolve a range of new challenging demanding substituents (e.g., propyl group) cause a dramatic
racemic sulfoxides with high efficiency including the decrease of the reaction rate. Although we were able to obtain
pharmaceutically relevant albendazole sulfoxide. The simplicity and valuable chiral synthons in a practically enantiopure form, the
the level of throughput is suitable for the future screening of substrate limitations prevent the method from utilizing more
metagenomic libraries for the discovery of new enzymes with complicated and pharmaceutically relevant chiral sulfoxides,
similar reactivities.
such as Armodafinil and Albendazole-(R)-sulfoxide).17–20 In order
to deal with this limitation we resolved to perform the directed
evolution of wild type MsrA. In spite of the fact that 3D NMR
structure of MsrA (E. coli) is known,21 we aimed at the
development of a high-throughput assay that would allow a
general and unbiased screen of a large sequence space of the
library of MsrA random mutants rather than focused site-
directed mutagenesis. Such a method would be applicable for
evolution of enzymes with similar reactivities without a priori
knowing the 3D structure or the positioning of the active site.
Furthermore, in the reverse mode, it could serve for a quick
active site mapping of sulfoxide reductases of unknown
structure.
The application of enzymes as biocatalysts for preparation
of high value chemicals and pharmaceuticals has experience a
rapid development in the past decades.1–3 The progress of the
field was enabled by inception of techniques of recombinant
DNA and DNA amplification.4,5 These technologies allowed for
the first time to obtain a large variety of enzymes from different
sources in sufficient quantities and purity. The next
breakthrough in the field of biocatalysis was the development
of the methods of directed evolution that were pioneered by
Arnold and others in the early 1990s.6–13 Utilization of DNA
libraries of given enzymes enables a researcher to create
millions of variants of a catalyst within a few days, which is in its
efficiency unsurpassed by development of chemical catalysts. In
the following screening process, the functional promiscuity of
the enzyme variants enables the new reactivities to be selected.
However, in spite of the success of the method, the screening
process still remains a significant bottle neck of this whole
enzyme evolution approach.
Herein, we report on the development of high-throughput
fluorogenic assay for directed evolution of sulfoxide reductases.
We have previously developed an efficient method for
deracemization of chiral sulfoxides,14 which utilizes natural
enzyme methionine sulfoxide reductase A (MsrA) for the kinetic
resolution of sulfoxides.15,16 We were thus able to deracemize a
Department of Organic Chemistry, Faculty of Science, Charles University in Prague
Hlavova 2030/8, 12843 Prague 2, Czech Republic. E-mail: misek@natur.cuni.cz
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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buffer solution of the probe was simply sprayed on the plates
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with colonies; a strong glow of those colDoOniI:e1s0t.1h0a3t9/oDv0eCrCe0x1p6r6e0sHs
wt MsrA was observed. By contrast, E. coli colonies without the
plasmid for wt MsrA overexpression showed no marked
increase of fluorescence with methyl GreenOx, which proved
the feasibility of the assay. In the next step, the wt MsrA gene
was diversified by error-prone PCR and the resulting library was
cloned and transformed into electrocompetent E. coli cells. The
presence of mutations in the gene (on average 3 random
mutations per gene) was confirmed by sequencing. For the
proof-of-principle experiment, five plates with roughly 20 000
colonies with mutant MsrAs were obtained. Then, the plates
were homogenously sprayed with the solution of propyl
GreenOx. After 30 min incubation at room temperature, five
colonies developed a significant yellow fluorescence (Figure 1).
These were harvested, re-plated and re-assayed. Two of the five
clones developed a strong yellow fluorescence after only 10
minutes. These clones termed clone 1 and clone 2 were further
analysed.
O-
O-
S+
wt MsrA (0.1 mol%)
+
S+
S
R1
rac-1
R2
R1
R2
R1
R2
50 mM PBS, pH 7.5
DTT (4 eq.)
(R)-1
2
37 °C, 24 h
O-
S+
O-
S+
O-
S+
(R)-1a
conv. 50%
ee >99%
(R)-1c
conv. <2%
ee -
(R)-1b
conv. 50%
ee >99%
Scheme 1 Kinetic resolution of sulfoxides with wild-type MsrA and its substrate
limitations.
Sequencing of the clones showed that clone 1 contains
mutations R18C, F52L and Q109H and clone 2 mutations F52L,
S106N and Q157V. Strikingly, both clones share the same amino
acid mutation of phenylalanine 52 to leucine that was however
caused by a different mutation in the same codon (clone 1: TTC
to CTC, clone 2: TTC to TTA). We expressed both clones and
measured the kinetics of reduction with propyl GreenOx. As
compared to the wild type MsrA, both clones showed a dramatic
increase in the reactivity (see ESI). Virtually the same kinetics of
both clones led us to a hypothesis that the mutation F52L might
be the major determinant of the increased reactivity.
Phenylalanine 52 is located directly in the active site of the
enzyme and creates a hydrophobic wall. Thus, its mutation to
smaller but still hydrophobic leucine might provide more space
for accommodation of bulkier substituents on the sulfoxide
moiety (R2), while not interfering with protein folding. In order
Typically, the screening approaches with highest throughput
in directed evolution of enzymatic activity involve chromogenic
or fluorogenic substrates that allows rapid and efficient
evaluation of the individual variants.22 However, there is only a
handful of such substrates and these allow to screen only a
limited
number
of
reactivities
(most
commonly
hydrolysis).23,24Thus, we utilized our expertise in the
development of fluorogenic probes for sulfoxide reductases and
designed a new, highly sensitive probe we call propyl GreenOx
(Scheme 2).25,26 The probe is synthesized in two simple steps and
shows negligible fluorescence (fl <0.001). However, upon
reduction of the sulfoxide moiety to sulfide, a marked increase
of the fluorescent signal is observed (fl = 0.046, max = 545 nm)
in both aqueous and non-aqueous environment. It has to be
noted that during the preparation/submission process of this
article another lab has reported similar fluorescent probes.27
O
O-
S+
S
S
reduction
N+
N+
TFA-
TFA-
non-fluorescent
fluorescent
gene library of MsrA
Propyl GreenOX (fl < 0.001)
sulfide 3a (fl = 0.046)
with random mutations
S
Scheme 2 A fluorescent probe design for sensing the activity of sulfoxide
reductases.
Fig. 1 A schematic representation of a high-throughput assay for the directed
evolution of MsrA. The left part shows a snapshot of a real plate from the
selection.
Given the substrate scope of natural MsrA, we hypothesized
that propyl GreenOx would be a good substrate for the directed
evolution of the enzyme. Indeed, when tested with the wild type
MsrA (0.5 mol%), the reactivity at four hours was negligible (see
SI). Also, methyl GreenOx variant of the probe was prepared and
tested with wt MsrA. As expected, a strong fluorescent signal
was observed, since the wt MsrA efficiently reduces the methyl
sulfoxide moiety. In addition, the probe was found to be well
permeable to E. coli cells, which makes it favourable for a simple
assay development. Therefore, we tested methyl GreenOx on
the E. coli colonies overexpressing wt MsrA on agar plates. The
to test this hypothesis, we created a clone with a single
mutation F52L by site-directed mutagenesis. This clone showed
basically the same kinetics of reduction of propyl GreenOx as
the clone 1 and 2, which confirmed the hypothesis that the F52L
mutation is solely responsible for the change in the activity of
the enzyme.
Next, we tested the wt MsrA and the clone F52L in reactions
with substrates 1a-1g (Table 1). While wt MsrA expectedly
resolved only substrates 1a and 1b with methyl and ethyl
substitution, the clone F52L was able to successfully resolve also
2 | J. Name., 2012, 00, 1-3
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substrates 1c and 1d with significantly larger propyl and butyl indeed was the case. Nevertheless, the new assayVcieawnAbrtiecleeOansliinlye
substituents in the sulfoxide moiety. Also, the challenging adjusted to implement a screening for enaDnOtI:io10se.1l0e3c9t/iDv0itCyCa0s1w66e0lHl.
substrate 1e with two bulky alkyl groups on the sulfoxide was As we have previously demonstrated, utilization of an
successfully resolved with an excellent enantioselectivity by enantiomerically pure fluorogenic probe with the non-
using the F52L clone. Delightfully, also the pharmaceutically fluorogenic competitor of the opposite chirality can easily
relevant albendazole-(R)-sulfoxide (1g) that is the active address this issue.28 As some sulfoxide reductases can act also in
metabolite of the essential anti-parasitic agent albendazole, can the reversed fashion as oxidases,29,30 this simple assay would
now be obtained with high enantioselectivity (94% ee). also be applicable for evolution of enzymes for enantioselective
Branching at the alpha carbon of the sulfoxide moiety (1f) seems oxidation of prochiral sulfides. In general, a similar approach can
to set the limits to the significantly expanded substrate scope of be applied for evolution of any enantioselective enzymes that
the F52L clone.
can catalyse both forward and reverse reactions (e.g.,
ketoreductases and transaminases, etc.).
Table 1 Substrate scope of kinetic resolution with wt MsrA and F52L MsrA mutant.[a]
In conclusion, we have developed a new simple and widely
applicable high-throughput screening assay for the directed
evolution of enantioselective sulfoxide reductases. In the proof-
of-principle experiment we have evolved natural MsrA. The
resulting mutant MsrA has a significantly broader substrate
scope, enabling to resolve a range of challenging chiral sulfoxide
with high efficiency and enantioselectivity, including the
pharmaceutically relevant albendazole-(R)-sulfoxide (1g). The
simplicity and high-throughput of the assay enables efficient
screening of large libraries of enzyme mutants with this
enantioselective reductase activity. Moreover, the level of
throughput makes this method also suitable for screening of
large metagenomic libraries for new enantioselective sulfoxide
reductases.
Entry
Product
wt MsrA
conversion
(ee)
F52L MsrA
conversion
(ee)
O-
S+
1
50%
(99%)
50%
(99%)
1a
O-
S+
2
3
50%
(99%)
50%
(99%)
1b
O-
S+
We acknowledge support of the Czech Science Foundation (GACR
17-25897Y). Also, we would like to thank Prof. Pavel Kočovský for
critical reading of the manuscript.
<3%
50%
(99%)
1c
O-
S+
Conflicts of interest
There are no conflicts of interest.
4
5
<3%
<3%
<3%
50%
(99%)
1d
O-
S+
Notes and references
50%
(99%)
1
2
3
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PBS (0.5 mL), 24 h. [b] 1.2 mol% of MsrA used.
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