Engineering strictosidine synthase: Rational design of a small, focused circular permutation library of the β-propeller fold enzyme

Article abstract of DOI:10.1016/j.bmc.2014.06.023

Strictosidine synthases catalyze the formation of strictosidine, a key intermediate in the biosynthesis of a large variety of monoterpenoid indole alkaloids. Efforts to utilize these biocatalysts for the preparation of strictosidine analogs have however been of limited success due to the high substrate specificity of these enzymes. We have explored the impact of a protein engineering approach called circular permutation on the activity of strictosidine synthase from the Indian medicinal plant Rauvolfia serpentina. To expedite the discovery process, our study departs from the usual process of creating a random protein library, followed by extensive screening. Instead, a small, focused library of circular permutated variants of the six bladed β-propeller protein was prepared, specifically probing two regions which cover the enzyme active site. The observed activity changes suggest important roles of both regions in protein folding, stability and catalysis.

Full text of DOI:10.1016/j.bmc.2014.06.023

Bioorganic & Medicinal Chemistry 22 (2014) 5633–5637
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Engineering strictosidine synthase: Rational design of a small,
focused circular permutation library of the b-propeller fold enzyme
c,
Eva Fischereder a, Desiree Pressnitz a,b, Wolfgang Kroutil a, , Stefan Lutz
⇑
⇑
a Department of Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
b ACIB GmBH, c/o, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
c Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Strictosidine synthases catalyze the formation of strictosidine, a key intermediate in the biosynthesis of a
large variety of monoterpenoid indole alkaloids. Efforts to utilize these biocatalysts for the preparation of
strictosidine analogs have however been of limited success due to the high substrate specificity of these
enzymes. We have explored the impact of a protein engineering approach called circular permutation on
the activity of strictosidine synthase from the Indian medicinal plant Rauvolfia serpentina. To expedite the
discovery process, our study departs from the usual process of creating a random protein library,
followed by extensive screening. Instead, a small, focused library of circular permutated variants of the
six bladed b-propeller protein was prepared, specifically probing two regions which cover the enzyme
active site. The observed activity changes suggest important roles of both regions in protein folding,
stability and catalysis.
Received 19 May 2014
Revised 9 June 2014
Accepted 11 June 2014
Available online 19 June 2014
Keywords:
Bioorganic chemistry
Biocatalysis
Circular permutation
Beta propeller protein
Protein engineering
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
thought to form a cap over the enzyme’s catalytic center. The active
site itself is located in the center of the protein structure with
Strictosidine synthases are part of the monoterpenoid indole
alkaloid biosynthetic pathway in higher plants leading to diverse
alkaloid products including high-value, active pharmaceutical
compounds used in the treatment of human diseases, such as can-
cer, malaria or schizophrenia.1 The enzymes are categorized as
amine lyases and catalyze a Pictet–Spengler-type condensation
between tryptamine (1) and the aldehyde secologanin (2) which
leads to the formation of (S)-strictosidine (3) (Fig. 1).2
Structurally, strictosidine synthases belong to the b-propeller
fold family characterized by an all-b architecture with four to eight
blades, each consisting of four antiparallel b-sheets, arranged
around a pseudo symmetry axis.3–8 The strictosidine synthase from
Rauvolfia serpentina (STR) is a six-bladed b-propeller protein (Fig. 2)
and represents one of the more extensively studied enzymes in this
group of lyases.1,9–12 Beyond its canonical fold, STR contains three
His307 and Glu309 as the key catalytic residues and multiple hydro-
phobic amino acids to line the substrate binding pocket.13
The ability of strictosidine synthase to catalyze carbon–carbon
bond formation, as well as its role in preparing a key intermediate
for a large family of pharmacologically interesting molecules
makes the enzyme an attractive target for bioorganic chemistry.
However, the broader application of the biocatalyst in combination
with unnatural substrates and analogs is complicated by the
enzyme’s high substrate specificity. Despite of the rather generic
hydrophobic binding pocket, STR exhibits very low tolerance for
amine and aldehyde analogs.1 One strategy to overcome these
limitations is the use of protein engineering. Stockigt and cowork-
ers recently reported two studies, employing site-directed
mutagenesis to introduce amino acid substitutions in four key
positions in the enzyme’s active site.12,14 Besides probing the role
of the catalytic residues His307 and Glu309, initial efforts to remo-
del the hydrophobic binding pocket were undertaken by changes
to Tyr151 and Val208. Separately, Bernhardt et al. developed a
plate-based screening assay for evaluating the catalytic activity
of STR variant with natural and unnatural substrates.15
helical segments (a1–a3). The first two helices are located in the
loop connecting blade-1 and 2 and are linked by a disulfide bridge
known to contribute to overall protein integrity. The third helix is
part of blade-3 and, together with the loop region in blade-5, is
In the present study, we have explored the effects of circular
permutation (CP) on the activity of STR. During CP, the native ter-
mini of a protein are linked with a short, flexible peptide linker and
new termini are introduced elsewhere in the protein sequence
⇑
Corresponding authors. Tel.: +43 316 380 5350; fax: +43 316 380 9840 (W.K.);
tel.: +1 404 712 2170; fax: +1 404 727 6586 (S.L.).
E-mail addresses: wolfgang.kroutil@uni-graz.at (W. Kroutil), sal2@emory.edu
(S. Lutz).
http://dx.doi.org/10.1016/j.bmc.2014.06.023
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

5634
E. Fischereder et al. / Bioorg. Med. Chem. 22 (2014) 5633–5637
E. coli DH5a strains were obtained from Invitrogen (Karlsruhe, Ger-
many). QIAquick Gel Extraction Kits and Miniprep Kits from Qiagen
(Hilden, Germany) were used for DNA isolation and purification.
Restriction enzymes NdeI, XhoI and SpeI, as well as Phusion-DNA
polymerase and T4 DNA ligase were obtained from New England
Biolabs (Frankfurt/Main, Germany). Taq-DNA polymerase was
obtained from Peqlab (Polling, AT). All other reagents were pur-
chased from Sigma–Aldrich (Steinheim, Germany), Merck (Darms-
tadt, Germany), Roth (Karlsruhe, Germany) and Oxid (Hempshire,
England). Enzyme graphics were prepared with PyMOL.25
Figure 1. Natural biotransformation catalyzed by strictosidine synthase (STR). The
condensation of tryptamine (1) and secologanin (2) produces the (S)-isomer of
strictosidine (3) with high enantioselectivity.
2.2. Construction of STR tandem gene sequence
The tandem STR gene sequence that served as template for the
preparation of circular permutated STR (cpSTR) variants was
assembled in pET17b using a truncated version of the STR gene
from Rauvolfia serpentina. Briefly, the N-terminal 28 amino acid
residues in STR which constitutes the native signal sequence, as
well as the unstructured 11 amino acids at the C-terminus were
deleted, leaving a 915-nucleotide truncated version of STR. The
tandem gene sequence was assembled by two separate PCRs; the
first truncated STR gene was amplified with primer set #1:
50-CGC CAT ATG CCG ATT CTG AAA GAA ATT CTG ATT-30 and 50-
CAG AAT CGG ACT AGT TCC ATA CAC CAG AAT ACC AAC GCT
ACC-30. The extra tri-nucleotide (–TCC–) together with the SpeI site
(underlined) in the reverse primer also encoded for the three-
amino acid peptide linker (-Gly-Thr-Ser-) needed to connect the
protein’s native termini. The second truncated STR gene was
obtained with primer set#2: 50-CTG GTG TAT GGA ACT AGT CCG
ATT CTG AAA GAA ATT CTG ATT-30 and 50-CGC CTC GAG ATA CAC
CAG AAT ACC AAC GCT ACC-50. The two PCR products were subse-
quently cloned into pET17b, initially using the NdeI/SpeI sites (PCR
product from reaction with primer set#1), followed by SpeI/XhoI
sites (PCR product from reaction with primer set#2). The correct
gene sequence was confirmed by DNA sequence analysis of the
plasmid.
Figure 2. Overview of strictosidine synthase (STR) structure from Rauvolfia
serpentina with secologanin (2) bound in the active site. (PDB codes: 2FP9,
2FPC).12 The six blades constituting the b-propeller fold are indicated in distinct
colors. The amino and carboxyl termini of the truncated STR (residues Pro85 to
Tyr333) are located in blade 6 and measure a distance of 7.2 Å. Based on their
proximity to the bound substrate,
a-helix 3 (amino acid positions 171 to 184)
which connects blades 2 and 3, as well as the extended loop (amino acid positions
271 to 279) in blade 5 were chosen as locations for new protein termini in our
rational CP experiment.
2.3. Preparation of selected cpSTR variants
through peptide backbone cleavage.16 Previous engineering efforts
by this approach have shown that the relocation of the protein ter-
mini can result in substantial activity gains.17–22 These functional
improvements could be traced back to enhanced accessibility of
the catalytic site and increases in local protein dynamics.23,24 The
latter effect can be particularly effective if the targeted region is
involved in rate-limiting conformational changes. In the specific
case of STR, CP seems a promising approach as B-factor analysis
With the help of the tandem STR template, selected cpSTR vari-
ants could be generated by simple PCR using site-specific primers
(see Table S1). All cpSTR PCR products were purified and digested
with NdeI and XhoI for subcloning into pET14b. Following confir-
mation of the correct DNA sequence, plasmids were transformed
into E. coli BL21(DE3)pLysS for heterologous protein expression.
of crystal structure data (PBD access codes: 2FP8 & 2FP912
)
2.4. Heterologous overexpression of cpSTR variants
indicates increased flexibility in two regions near the active site.
More specifically, the two regions correspond to the cap domains
E. coli host cells containing one of the pET14b/cpSTR variants
were cultured in 2-YT-media supplemented with ampicillin
consisting of
a3 in blade-3 and the loop in blade-5. Even though
there is no direct experimental evidence in support of a rate-
limiting conformational change during catalysis involving these
two cap domains, we chose to explore the effects of CP on these
two regions.
(100 lg/mL) and chloramphenicol (34 lg/mL) overnight. Aliquots
were used to inoculate fresh culture media and cells were incu-
bated at 37 °C until cell density reached an OD600 of approximately
0.6–0.8. Protein expression was started by addition of IPTG (final
concentration: 0.6 mM) and incubation at 20 °C was continued
for 24 h. Cells were harvested by centrifugation and pellets were
washed once with buffer (50 mM PIPES, pH 6.8). Finally, cells were
resuspended in 15 mL PIPES buffer and lyophilized. Freeze-dried
cell pellets were stored at 4 °C until further use.
2. Material and methods
2.1. Materials
The codon-optimized gene sequence from Rauvolfia serpentina
(STR) (GeneBank: Y00756.1) was ordered from Life Technologies
(Darmstadt, Germany). Oligonucleotide primers were obtained
from IDT (Coralville, IA) and Eurofines MWG operon (Ebersberg,
Germany). Expression vectors pET14b and pET17b were purchased
from Novagen (Darmstadt, Germany). Escherichia coli strain
BL21(DE3)pLysS, as well as chemical competent E. coli Top10 and
2.5. Enzyme assay
In a typical assay to determine enzymatic activity of wild type
STR and individual cp variants, 0.3–2.5 mg of lyophilized cells were
rehydrated in 225
lL PIPES buffer (50 mM, pH 6.8) for 30 min at
28 °C. Next, 25 L of substrate stock solution (10 mM tryptamine
l

E. Fischereder et al. / Bioorg. Med. Chem. 22 (2014) 5633–5637
5635
hydrochloride, 20 mM secologanin (Sigma–Aldrich, Vienna)) was
added and samples were placed on a mechanical shaker at 28 °C.
Aliquots were removed from the reaction mixture after 5, 10 and
20 min, as well as after 1, 3 and 24 h. The reactions were quenched
truncated STR gene in a front-to-tail fashion (Fig. 3).22 Linked by
a 9-bp element encoding the three-amino acid linker, the tandem
construct served as template for subsequent PCR amplification of
selected CP variants with site-specific oligonucleotide primers. Ini-
tial attempts to assemble the tandem genes via simple PCR overlap
extension were unsuccessful, so stepwise cloning of the two STR
gene copies into pET-17b via NdeI/SpeI (for STR copy 1) and SpeI/
XhoI (for STR copy 2) was employed to generate the PCR template.
The rational design of selected CP variants focuses on helical
with 250
buffer (30 mM, pH 2.8). The solution was centrifuged (5 min,
13,000 rpm) and 50 L aliquots were subjected to HPLC analysis
(Agilent; LUNA C18 column (Phenomenex, 1.24 ꢀ 45, 5 m); flow
lL methanol, followed by addition of 250 lL NH4-formate
l
l
rate = 1 mL/min, absorption measured at 280 nm) using solvent A:
acetonitrile/0.1% TFA and solvent B: NH4-formate (30 mM, pH 2.8)
and a linear gradient from 10% A to 50% A over 8 min, then to 80%
A in 3 min and finally back to 10% A in 0.5 min, followed by re-equil-
ibration for 3.5 min. The retention times for substrates and product
were as follows: tryptamine = 7.43 min; secologanin = 7.97 min;
strictosidine = 9.28 min. Product identification based on reference
sample of chemically synthesized (R/S)-strictosidine.
region
a3 in blade 3 and an extended loop in blade 5 (Fig. 2,
inserts). These two segments of the protein are located near the
enzyme active site and are believed to be part of the substrate
entrance tunnel in STR. Previous CP studies on a number of other
proteins suggest that such regions are particularly promising as
conformational changes in these portions of the protein structure
are often linked to catalytic turnover, yet changes do not directly
perturb the critical topology of active site residues.17,19,22,23 To
probe these regions, three cpSTR variants with new termini in
a3
3. Results and discussion
were created, cleaving the peptide backbone at residues Tyr171,
Ile179, and Asp184. While positions 171 and 184 flank the helical
segment, position 179 is located in the center part of the helix. Sep-
arately, four cpSTR variants with termini relocated in positions
Leu272, Gly274, His277, and Arg279 in the blade-5 loop structure
were prepared. All seven variants were obtained by PCR amplifica-
tion, using gene-specific primers (Table S1) and plasmid containing
the tandem STR gene construct as template. The PCR products were
subcloned into pET-14b for subsequent heterologous expression in
E. coli cells. Overexpression of cpSTR variants was evaluated over a
range of induction temperatures and times, as well as IPTG concen-
trations. The four variants with relocated termini in the blade-5
loop consistently yielded soluble protein (Fig. S1). In contrast, the
three variants carrying new termini in the helical region of
blade-3 failed under all tested conditions to produce soluble pro-
tein and instead only led to aggregate formation. A possible expla-
In preparation for the CP experiment, the amino and carboxy
ends in the native STR sequence were trimmed to allow for a short
peptide linker to connect the original protein termini. The full-
length STR includes a 28 amino acid-long N-terminal cellular
export sequence and an 11-residue C-terminal unstructured
region. Both regions are invisible in the crystal structure of
R. serpentina (PDB codes: 2FP8/912) and are considered irrelevant
to the catalytic function of STR. The absence of changes in catalytic
performance of truncated STR compared to wild type STR was
verified experimentally (Table 1). The truncation of these residues
generates a shortened wild type STR with Pro29 as the new N-ter-
minus and Tyr333 as the C-terminus. As shown in Figure 2, the two
new termini are both located on blade 6 in close proximity to each
other. The distance of 7.2 Å between them can be bridged by a 3–4
amino acid linker. Glycine-rich linkers of six or less residues pro-
vide maximum flexibility to avoid conformational constraints
while minimizing protein destabilization through extended loop
formation.16 We therefore chose the -Gly-Thr-Ser-tripeptide as
connector for truncated STR. Besides the flexibility of Gly and the
hydrophilicity of the Ser and Thr side chains, the corresponding
gene sequence also encodes a unique SpeI restriction site which
is helpful in subsequent subcloning steps.
nation for sensitivity of the
a3 region to CP could be its critical
involvement in the protein’s folding pathway. Furthermore, exam-
ples in the literature have demonstrated that the cleavage of the
peptide backbone increases local conformational flexibility, an
The 924-bp gene encoding the truncated STR (305 + 3 amino
acids) was used as template for the CP experiment. Rather than
creating a large random CP library, we were interested in creating
a small, focused set of enzyme variants with termini relocations in
specific regions within the protein structure. The strategy would
enable a quick and effective probing of the most promising regions
near the active site for potentially beneficial effects on catalytic
performance. For generating these specific CP variants, we there-
fore created a tandem gene construct, fusing two copies of the
Table 1
Enzymatic activity of native and truncated STR, as well as CP variants for conversion
of tryptamine (1) and secologanin (2)
cpSTR variant
U (l
mol/min/mg ꢀ 10ꢁ4
)
RA (%)
Wild type STR
Truncated STR
cpSTR273
cpSTR274
cpSTR275
cpSTR276
cpSTR277
cpSTR278
cpSTR279
70
67
8
8
8
8
4
8
8
100
95
12
12
12
12
6
Figure 3. Strategy to prepare gene sequences encoding for selected circular
permutation variants of Rauvolfia serpentina strictosidine synthase (STR). Deletion
of the initial 84 nucleotides from the full-length gene eliminated the N-terminal 28-
amino acid signal sequence while truncation of the last 36 nucleotides removed the
C-terminal 11 amino acids (plus STOP codon). Two copies of the truncated STR,
fused by a 9-nucleotide linker, were cloned back-to-back and the resulting tandem
sequence served as template for PCR amplification of selected cpSTR variants with
the help of gene-specific primers. Individual gene variants were cloned into pET-
14b for expression of the corresponding protein products.
12
12
Activity reported in absolute units (U) and percent of residual activity (RA).

5636
E. Fischereder et al. / Bioorg. Med. Chem. 22 (2014) 5633–5637
effect that can be highly detrimental to overall protein
structure.26,27
enzyme is characterized by its limited tolerance for substrate
analogs, a highly undesirable property in light of the enzyme’s
application in biosynthesis of strictosidine derivatives. Enhanced
substrate promiscuity as a result of increased conformational
flexibility and structural perturbation of the active site are well
established effects.
Preliminary screening of soluble cpSTR variants indicated enzy-
matic activity for three of the four blade-5 variants. After 24-h reac-
tion time, cpSTR274, 277 and 279 showed complete conversion of 1
and 2 while cpSTR272 had no detectable activity. These promising
findings encouraged us to create a second set of permutants, cover-
ing five additional positions in the loop region (residues Glu271,
Asp273, Asn275, Met276 and Gly278; Fig. 2 insert). With the excep-
tion of cpSTR271, these second-generation variants also exhibited
catalytic activity in prescreening. The precise cause for the inactivity
of cpSTR271 and 272 is unclear. Although both proteins could be
overexpressed in soluble form and cpSTR273, their immediate
neighbor, shows fine catalytic activity, multiple attempts to detect
substrate conversion in these two variants were unsuccessful.
More precise measurements of enzyme activity of the seven
active cpSTR variants were subsequently performed by collecting
multiple time points over the course of the 24-h reaction. Aliquots
of reaction mixture were quenched by addition of methanol and
product formation was quantified by HPLC analysis. Under our
experimental conditions, wild type STR completed substrate
conversion within 30 min while equivalent amounts of the
engineered enzymes showed noticeably slower rates of formation
of native product 3 (Fig. 4). Nevertheless, all enzyme variants
resulted in 100% substrate conversion after 24-h as observed in
the prescreening assays. Based on these data, the calculation of
units of enzyme activity showed that the engineered biocatalysts
possess between 6% and 12% residual activity compared to wild
type STR (Table 1). Enzyme stability was deduced from the time
course of the reaction to complete conversion after 24 h, suggest-
ing that the decline in activity is not caused by destabilization of
the engineered proteins. The findings rather suggest that increased
conformational flexibility and altered protein dynamics in the loop
region of blade-5 have a detrimental effect on STR catalysis. Previ-
ously published results from a small-scale mutagenesis study to
explore the key catalytic residues in STR reported a quite signifi-
cant increase in the binding affinity for 2 upon substitution of
Ala for His in position 307, a residue located in a flanking loop
which connects blades 5 and 6.12 Perturbation of the neighboring
loop (residues 271–279) could easily interfere with the proper
topology of His307, in turn resulting in the observed decline in
activity due to lower substrate binding affinity. As part of ongoing
and future experiments, a more comprehensive analysis of the
kinetic parameters for selected cpSTR variants will be insight-
ful for rationalizing the functional changes observed in
these engineered enzymes. Separately, we are curious to explore
the effects of CP on the substrate specificity of STR. The native
Beyond the immediate functional consequences of CP on the
two target regions (a3 in blade-3 and loop in blade-5) in STR, the
present work also highlights one of the nagging problems with
focused library design. While our current site selection was driven
by active site proximity and the nature of these particular structure
elements, the question remains whether or not the right regions
were chosen for beneficial functional change. In particular two
segments in the STR structure including the connecting loop
between blades 6 and 1, as well as the extended helical region
between blades 1 and 2 will be of interest in future studies. In gen-
eral, a systematic evaluation of all possible permutants, although
more laborious, may outweigh the uncertainty associated with
focused, regional libraries due to limited predictability of beneficial
termini relocation sites.
Finally, the results from engineering STR may also reflect an
emerging broader theme in redesigning proteins by CP. Strictosi-
dine synthase, xylanase from Bacillus circulans as well as red and
green fluorescence proteins (FPs) from Aequorea victoria and Disco-
soma coral, respectively, have all recently been studied by this
engineering approach.19,28,29 In all four examples, the resulting
variants show at best moderate functional gains which is in sharp
contrast to some of the catalytic enhancements observed in other
enzymes.17,22 A possible rationale for the poor response of these
proteins to engineering by CP could be found in their shared
topologies, all consisting of b-sheet rich protein scaffolds. STR is
a member of the b-propeller fold family, the core structure of the
Bacillus xylanase is a b-sandwich domain, and the FPs consist of
eleven-stranded, cylindrical b-barrels. Previous computational
and experimental studies have pointed out the conformational
restrictions in b-sheet structures, resulting from cooperative stabil-
ization of individual b-strands which translates into limited local
flexibility in these scaffolds.30,31 As such, potential functional
benefits due to altered flexibility upon peptide backbone cleavage
during circular permutation might be limited in b-rich proteins.
4. Conclusions
In this study, a small focused circular permutation library of
strictosidine synthase was generated by rational design and evalu-
ated for its catalytic function. The work marks the first example for
engineering a member of the b-propeller fold family via this
approach and yielded several active variants albeit with inferior
catalytic performance for the native substrates. Nevertheless, the
protein does tolerate termini relocation without loss of overall
structural integrity in one of the two regions explored in this study.
These results not only justify further studies to assess the impact of
CP in the remaining loops near the active site but also offer oppor-
tunities for novel enzymes upon combination with mutagenesis
experiments. STR belongs to a class of enzymes still rarely investi-
gated by protein engineers. Our findings represent a first step
towards identifying possible essential polypeptide segments
beyond the immediate active site residues. Finally, the enhanced
local protein flexibility near the active site promises enhanced
substrate promiscuity, an invaluable property for employing the
biocatalyst in the efficient synthesis of strictosidine analogs.
Acknowledgments
Figure 4. Time course for strictosidine (3) synthesis catalyzed by truncated STR
(tSTR) and CP variants. While the CP variants exhibit low specific activities than
tSTR, extended reaction times of 24 h resulted in 100% conversion of substrate with
all tested enzymes.
The authors would like to thank the members of the Kroutil and
Lutz labs for their helpful comments and suggestions during

E. Fischereder et al. / Bioorg. Med. Chem. 22 (2014) 5633–5637
5637
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Supplementary data (specific primer oligonucleotide sequences,
protein sequences of cpSTR variants, and protein expression data)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2014.06.023.
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