Substrate specificity of strictosidine synthase

Article abstract of DOI:10.1016/j.bmcl.2006.01.098

Strictosidine synthase catalyzes a Pictet-Spengler reaction in the first step in the biosynthesis of terpene indole alkaloids to generate strictosidine. The substrate requirements for strictosidine synthase are systematically and quantitatively examined and the enzymatically generated compounds are processed by the second enzyme in this biosynthetic pathway.

Full text of DOI:10.1016/j.bmcl.2006.01.098

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2475–2478
Substrate specificity of strictosidine synthase
Elizabeth McCoy, M. Carmen Galan and Sarah E. O’Connor*
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA
Received 6 January 2006; revised 20 January 2006; accepted 20 January 2006
Available online 14 February 2006
Abstract—Strictosidine synthase catalyzes a Pictet–Spengler reaction in the first step in the biosynthesis of terpene indole alkaloids
to generate strictosidine. The substrate requirements for strictosidine synthase are systematically and quantitatively examined and
the enzymatically generated compounds are processed by the second enzyme in this biosynthetic pathway.
Ó 2006 Elsevier Ltd. All rights reserved.
The terpene indole alkaloids are a structurally complex
class of alkaloids used for a variety of medicinal purpos-
es such as anti-cancer and anti-hypertensive agents. In
the first committed step of the terpene indole alkaloid
biosynthetic pathway, tryptamine (1) and secologanin
NH2
Strictosidine
Synthase
NH
H
O-Glc
1
N
N
H
O
1
H
H
3
O
O-Glc
O
MeO C
2
(
idine synthase through a stereoselective Pictet–Spengler
2) react to form strictosidine (3) by the enzyme strictos-
MeO C
2
Strictosidine
Glucosidase
2
2
–5
condensation (Scheme 1).
Strictosidine is further
modified by strictosidine glucosidase to yield an inter-
mediate that rearranges in vitro to form cathenamine
N
H
spontaneous
OH
NH
H
O
6
,7
N
(
Scheme 1).
H
O
N
MeO C
Cathenamine
2
H
MeO C
Strictosidine synthase was first isolated thirty years ago,
and the enzyme has been cloned, heterologously ex-
2
3
,2,8–15
Scheme 1. First committed steps of terpene indole alkaloid
biosynthesis.
pressed,
and structural analysis of this enzyme is
1
6
in progress. Although these biosynthetic enzymes
may provide an opportunity for production of novel
alkaloid derivatives, a firm understanding of the param-
eters involved in substrate recognition is needed to take
advantage of this complex pathway. A systematic and
quantitative analysis of strictosidine synthase substrate
specificity was therefore initiated.
kcat and over 100-fold variation in K . In all cases, prod-
m
uct formation was assessed by the appearance of a new
peak on an HPLC chromatogram that co-eluted with a
chemically synthesized (mixture of diastereomers) stan-
dard and displayed the correct molecular weight by
1
7
ESI mass spectrometry. Non-enzymatic reactions were
not observed during these assays, which were conducted
at neutral pH and micromolar to low millimolar sub-
strate concentrations.
Enzyme activity of strictosidine synthase (Catharanthus
roseus) was measured by HPLC, in which the amine
starting material disappearance and product formation
were monitored by UV. Km and kcat values were calcu-
lated for a selection of the most active amine substrates
using strictosidine synthase expressed in Escherichia coli.
This selection of substrates yielded a 40-fold variation in
18
Amine substrate specificity. Notably, strictosidine syn-
thase could synthesize alternative heterocyclic deriva-
tives, utilizing both the 3-(2-aminoethyl)-benzofuran
(
4) and benzothiophene (5) analogs, though at a dimin-
ished rate relative to the tryptamine 1 substrate.
Although the low activity of the thiophene substrate
precluded a quantitative comparison of 4 and 5, the
rate of reaction of benzothiophene 5 is significantly
Keywords: Alkaloids; Biosynthesis; Enzyme catalysis; Glycosidase;
Pictet–Spengler.
*
Corresponding author. Tel.: +1 617 324 0180; fax: +1 617 324
505; e-mail: soc@mit.edu
0
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.098

2
476
E. McCoy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2475–2478
Table 1. Substrates tested with strictosidine synthase
slower than that of benzofuran 4. The benzofuran 4
exhibits a Km value close to that of tryptamine, but dis-
plays a significantly reduced kcat, suggesting that the
electron-deficient nature of the benzofuran ring slows
catalysis (Table 2). N-Methyl tryptamine 6 (Table 1)
is not a competent substrate, suggesting that the en-
zyme tolerates only small steric perturbations at the in-
dole nitrogen.
Unnatural substrate
Strictosidine analog
NH
O-Glc
NH
2
X
H
X
4
5
X = O
X = S
O
2
MeO C
NH
2
No reaction
To further explore the effect of electron density on catal-
ysis, the indole ring was substituted with electron-with-
drawing substituents (fluoro = F) at each of the indole
ring positions (Tables 1 and 2). Substitution with a flu-
oro moiety results in a decrease in kcat in each case,
again suggesting that the enzymatic reaction is inherent-
ly dependent on the electron density of the amine sub-
strate. Although the kcat/Km ratios for all fluorinated
derivatives 7–10 are comparable, the Km for 4-fluoro-
substituted tryptamine 7 is approximately 3- to 5-fold
N
Me
6
4
4
R
R
5
5
R
R
4
5
NH
H
NH2
6
6
R
O-Glc
R
6
N
7
N
H
7 R4 = F; R5, R6, R7 = H
8 R5=F; R4, R6, R7 = H
7
7
R
H
R
O
9
1
R6=F; R4, R5, R7 = H
0 R7=F; R4, R5, R6 = H
MeO C
2
4
R
4
R
4
NH
H
O-Glc
higher than the K value for the other fluorinated deriv-
m
NH
2
N
7
77
N
H
11 R4 = Me; R7 = H
14 R7 =Me; R4 = H
R
H
atives. However, the kcat for the 4-fluoro derivative is
also significantly higher than those of the other fluoro-
tryptamine substrates, particularly those of the 5- and
O
R
2
MeO C
5
R
6
-fluoro derivatives (substrates 8 and 9). Interestingly,
5
6
NH
2
No reaction
6
the rate of the chemically catalyzed Pictet–Spengler
reaction between 4- or 7-fluorotryptamine (compounds
R
N
H
12 R5 = Me; R6 = H
13 R6 = Me; R5 = H
HO
7
or 10) and secologanin 2 is significantly slower than
that of the same reaction with 6- or 5-fluorotryptamine
compounds 8 or 9) (data not shown). Clearly, the posi-
HO
NH
O-Glc
(
NH2
N
H
H
tion of the electron-withdrawing group on the indole
ring has a significant effect on enzymatic catalysis, and
future experiments are directed at deciphering the rea-
sons behind this observation.
N
15
H
O
MeO C
2
X
NH
2
No reaction
No reaction
N
1
1
6 X = CH
7 X = N
H
Early qualitative studies with several tryptamine deriva-
tives indicated that limited substitution on the indole
NH
2
2
n
ring was tolerated. To systematically quantify the effect
of indole ring substitution on K and kcat, each position
1
8 n = 1
N
19 n = 3
m
H
of the indole ring was substituted with a methyl group
(
compounds 11–14, Table 1) and the kinetic parameters
NH
O-Glc
of active substrates were measured (Table 2). Reactivity
of substrates with methyl substitutions in the 4 (com-
pound 11) and 7 (compound 14) indole positions was ac-
tive, while substrates with substitutions at the 5
NH2
N
H
H
N
H
O
2
2
0
MeO
2
C
(
1
compound 12) and 6 (compound 13) positions (Tables
and 2) were not active (Fig. 1). The Km for the 4-
NH
2
No reaction
N
H
1
substituted tryptamine analog 11 was approximately 2-
fold lower than the Km for 7-substituted compound
R
H
O
1
6
4, while substrates with methyl moieties in the 5 and
positions—compounds 12 and 13—were completely
No reaction
O-Glc
O 22 R = t-butyl
inactive. Substitution with a hydroxyl group in the
position (compound 15, Tables 1 and 2) did yield an
MeO C
2
23 R = butyl
5
H
O
active substrate, though the Km was the highest mea-
sured in this series—a 60-fold increase compared to
the native substrate tryptamine. Therefore, strictosidine
synthase does not readily tolerate substitution at
positions 5 and 6 of tryptamine and is most tolerant
of substitutions at positions 4 and 7 (Fig. 1).
NH
H
O-Glc
O-Glc
N
H
O
24 R = ethyl
25 R = allyl
O
R
O
O
O
R
O
The 2-pyrrole-3-ethylamine analog (16) along with the
isosteric histamine (17) were not turned over by strictos-
idine synthase, indicating that the benzyl moiety is abso-
lutely required for recognition by the enzyme (Table 1).
It was previously established that tryptophan, phenyl-
ethylamine, and tyramine are not accepted by strictosi-
dine synthase, and since pyrrole substrates are also
not tolerated, we conclude that the basic indole
2

E. McCoy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2475–2478
2477
framework is required for recognition by this enzyme.
H
O
Interestingly, the only other sequenced ‘Pictet–Spengler-
ase’ (norcoclaurine synthase), which utilizes tyrosine
derived amine and aldehyde substrates, exhibits no
O-Glc
NH
2
O
X
RO C
2
1
9
sequence homology to strictosidine synthase.
Figure 1. Solid arrows highlight the positions most amenable to
substitution in the tryptamine and secologanin substrates for strictos-
idine synthase. Dashed arrows indicate positions for which compar-
atively weaker activity was observed.
Strictosidine synthase proved to have stringent require-
ments for the side chain of tryptamine, as evidenced
by the lack of turnover exhibited by 3-methylamine-in-
2
0
dole (18) and 3-propylamine-indole (19) (Table 1).
However, 2-(1-methyl)-ethylaminoindole (±-a-methyl-
tryptamine, 20, Table 1) yielded a small amount of prod-
uct. No product formation was observed with the
bulkier a-di-methyltryptamine (21) (Table 1).
All enzymatically generated strictosidine derivatives
(Table 1) were incubated with the second enzyme of
the pathway, strictosidine-b-glucosidase (C. roseus).
The disappearance of the strictosidine derivative (start-
ing material) peak by HPLC demonstrated that each
of the strictosidine derivatives was processed by strictos-
idine glucosidase. Additionally, when the 4-fluoro and
4-methyltryptamine substrates (compounds 7 and 11)
were incubated in the presence of secologanin 2 with
both enzymes, products correlating to the molecular
weight of cathenamine analogs were observed when ana-
lyzed by mass spectroscopy; cathenamine is the predict-
ed product of this deglycosylation reaction with
2
1
Aldehyde substrate specificity. To investigate the alde-
hyde substrate specificity of strictosidine synthase, two
of the key functional groups of the secologanin substrate
were modified and then utilized in enzyme assays.
A streamlined gram-scale isolation protocol of secolog-
anin from a local source of Lonicera tatarica enabled a
semisynthetic approach to yield secologanin deriva-
2
2
tives. Olefin cross-metathesis was used to introduce a
variety of alkyl groups at the vinyl position of secologa-
nin (i.e., compounds 22 and 23, Table 1). However,
these bulkier groups at the vinyl position completely
prevented turnover by strictosidine synthase (Table 1).
In contrast, trans-esterification at the methyl ester with
larger alkyl groups (compounds 24 and 25) gave sub-
strates that were turned over by the enzyme to yield
the corresponding strictosidine analogs, suggesting that
this is a more promising position for derivatization.
2
7
tryptamine and secologanin. While our data are con-
sistent with the formation of cathenamine analogs as is
6
reported in the literature, isolation and spectroscopic
characterization of these deglycosylated strictosidine
analogs will provide further structural confirmation.
These results suggest that the substrate specificities of
strictosidine synthase and glucosidase are sufficiently
complementary to work in tandem to produce a variety
of terpene indole alkaloid intermediate analogs.
While these results suggest that strictosidine synthase
can produce a range of strictosidine analogs, it remained
to be established whether these intermediates can be
processed by the downstream terpene indole alkaloid
machinery to produce novel, biologically active
alkaloids. In the next step of the pathway, a dedicated
glucosidase hydrolyzes the glycosidic linkage of strictos-
idine which rearranges in vitro to form cathenamine
This study systematically and quantitatively probes the
substrate scope of the indole amine substrate for the first
committed step of the terpene indole alkaloid pathway.
This enzyme catalyzes a Pictet–Spengler condensation,
which, although widely utilized in alkaloid biosynthetic
pathways, remains a largely unexplored enzymatic reac-
tion. Semisynthetic efforts have also yielded secologanin
derivatives that are turned over by strictosidine syn-
thase. The dedicated glucosidase for this pathway is able
to turnover strictosidine analogs generated by strictosi-
dine synthase, suggesting that strictosidine synthase
and strictosidine glucosidase can work in concert to
utilize ‘unnatural’ substrates to generate analogs of
alkaloid intermediates.
6
,7,23
(
Scheme 1).
Therefore, the first two enzymes con-
struct the basic 5-ring framework of the corynanthe
alkaloids. The corynanthe framework is an important
2
4–26
pharmaceutical scaffold,
and derivatization of these
molecules with substitutions on the indole or secologa-
nin moieties may result in interesting new compounds
with useful biological properties.
Table 2. Kinetic parameters for the most highly active amine
strictosidine synthase substrates
Acknowledgments
À1
À1 À1
)
Substrate
K
m
(lM)
k
cat (min
)
k
cat/K
m
(M /s
We gratefully acknowledge financial support from the
Smith Family Medical Foundation, the American
Chemical Society Petroleum Research Fund (41727-
G4), 3M, Amgen Inc, and MIT.
1
4
7
8
9
7.4
7.7
0.9
2030
50
0.023
0.35
0.043
0.056
0.11
0.19
0.29
0.096
42
139
101
105
141
40
7.1
8.9
10
11
14
15
13
80
Supplementary data
198
1200
24
1.3
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.01.098.
k
cat and K
m
were measured using a purified E. coli preparation of
strictosidine synthase.

2478
E. McCoy et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2475–2478
18. Compounds 11 and 13 were synthesized from the corre-
References and notes
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1
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2
2
1. Synthesis of these derivatives is described in Galan, M. C.;
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1
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methanol (200 mL). The slurry stood for 50 min and was
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6. Although a structure is not yet reported, the enzyme has
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7. The masses of product peaks that resulted from the
reaction of secologanin and the following amines were: 4
1
tography (CH Cl –CH OH, 9:1). Fractions containing
2
2
3
+
+
secologanin were pooled, concentrated, and the resulting
solid was further purified by C18 column chromatography
(H O–CH OH, 7:3) to yield purified secologanin (1 g).
2 3
NMR and mass spectral data matched previously reported
values.
(
[M+H] expect. 532.2, obsd 532.3); 5 ([M+H] expect.
+
48.2, obsd 548.3); 7 ([M+H] expect. 549.2, obsd 549.2); 8
5
(
+
[M+H] expect. 549.2, obsd 549.4); 9 ([M+H] expect.
+
+
49.2, obsd 549.2); 10 ([M+H] expect. 549.2, obsd 549.2);
5
+
1 ([M+H] expect. 545.2, obsd 545.2); 14 ([M+H]
+
1
expect. 545.2, obsd 545.4); 15 ([M+H] expect. 547.2,
+
2
2
2
2
2
3. Barleben, L.; Ma, X.; Koepke, J.; Peng, G.; Michel, H.;
Stoeckigt, J. Biochim. Biophys. Acta 2005, 1747, 89.
4. Roquebert, J.; Demichel, P. Eur. J. Pharmacol. 1984, 106,
+
obsd 547.4); 20 (both diastereomers) ([M+H] expect.
545.2, obsd 545.3). Product peaks that resulted from the
reaction of tryptamine and the following aldehydes were:
2
03.
+
4 ([M+H] expect. 545.3, obsd 545.7); 25 ([M+H]
+
5. Li, S.; Long, J.; Ma, Z.; Xu, Z.; Li, J.; Zhang, Z. Curr.
Med. Res. Opin. 2004, 20, 409.
6. Moreno, P. R. H.; Van der Heijden, R.; Verpoorte, R.
Plant Cell Tissue Org. Cult. 1995, 42, 1.
7. Substrate 7 ([M+H] expect. 369.4, obsd 369.5) and 11
2
expect. 557.2, obsd 557.2). Synthesis of authentic stan-
dards (diastereomeric mixtures) and evaluation of chem-
ical reaction rates was performed by reacting amine and
aldehyde substrates (20 mM concentration) under aque-
ous conditions (150 mM maleic acid, pH 2).
+
+
(
[M+H] expect. 365.4, obsd 365.5).