Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD+, NADPH, or NAD+/10-Oxogeranial: Reaction Mechanisms

Article abstract of DOI:10.1002/anie.201508310

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD⁺, NADPH or NAD⁺/10-oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 Oζ is positioned 2.5 ? from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4-C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis.

Full text of DOI:10.1002/anie.201508310

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International Edition: DOI: 10.1002/anie.201508310
German Edition: DOI: 10.1002/ange.201508310
Biosynthesis
Structures of Iridoid Synthase from Cantharanthus roseus with Bound
NAD+, NADPH, or NAD+/10-Oxogeranial: Reaction Mechanisms
Yumei Hu, Weidong Liu, Satish R. Malwal, Yingying Zheng, Xinxin Feng, Tzu-Ping Ko, Chun-
Chi Chen, Zhongxia Xu, Meixia Liu, Xu Han, Jian Gao, Eric Oldfield,* and Rey-Ting Guo*
Abstract: Structures of the iridoid synthase nepetalactol
synthase in the presence of NAD+, NADPH or NAD+/10-
oxogeranial were solved. The 10-oxogeranial substrate binds in
a transoid-O1-C3 conformation and can be reduced by hydride
addition to form the byproduct S-10-oxo-citronellal. Tyr178
Oz is positioned 2.5 Š from the substrate O1 and provides the
second proton required for reaction. Nepetalactol product
formation requires rotation about C1–C2 to form the cisoid
isomer, leading to formation of the cis-enolate, together with
rotation about C4–C5, which enables cyclization and lactol
production. The structure is similar to that of progesterone-5b-
reductase, with almost identical positioning of NADP, Lys146-
(147), Tyr178(179), and F342(343), but only Tyr178 and
Phe342 appear to be essential for activity. The transoid 10-
oxogeranial structure also serves as a model for b-face hydride
attack in progesterone 5b-reductases and is of general interest
in the context of asymmetric synthesis.
substrates or other ligands. The structures of the iridoid
synthases are of interest because their products are converted
into important natural products, compounds such as the
anticancer drug vincristine (2), as well as being of mechanistic
interest, not least because their amino acid sequences have
high homology to the plant progesterone (3) 5b-reductases
involved in the formation of cardiac glycoside drugs such as
digitoxin (4). A simplified version of the biosynthesis of
vincristine (2) from isopentenyl diphosphate (5) and dime-
thylallyl diphosphate (6) is shown in Scheme 1A; the
structures of other molecules discussed herein are shown in
Scheme 1B.
We expressed and purified an iridoid synthase, Canthar-
anthus roseus (1R, 4aS, 7S, 7aR) nepetalactol (7) synthase,
from the corresponding chemically synthesized gene (Gene-
bank accession number: K7WDL7.1). C. roseus is the
Madagascar periwinkle and produces the anticancer indole
terpenes vincristine (2) and vinblastine (8) from iridoids.
Attempts to crystallize full-length protein were unsuccessful,
so we attempted crystallization of two N-terminus-truncated
variants, DN13 and DN25. DN25 crystals were obtained
(details of protein expression, purification, and crystallization
are given in the Supporting Information). The activity of the
DN25 construct was (within experimental error) the same as
that of the wild-type protein (Figure S1 in the Supporting
Information). We then co-crystallized this IRIS with either
NAD+, NADPH, or NAD+ + 10-oxogeranial and solved the
structures. Full data acquisition and refinement details are
given in Table S1 in the Supporting Information.
We first obtained the structures of IRIS with either
NADPH or NAD+ bound. A stereo view of the NADPH
structure is shown in Figure 1A; the NAD+ structure is
virtually identical (a 0.21 Š root mean square deviation,
rmsd).The protein fold is most similar to that of progesterone
5b-reductase from Digitalis lanata, a plant that produces
cardiac glycosides such as 4. There is a 1.0 Š Ca rmsd over 351
residues (using the PDBe Fold Server[5]). An alignment of the
active sites of the IRIS protein (+ NADPH, PDB ID: 5DBF)
and the D. lanata 5b-reductase (+ NADP+, PDB ID code
2V6G[6]) is shown in Figure 1B. The two active-site structures
are very similar and interestingly, the Tyr179 shown by site-
directed mutagenesis to be essential for progesterone 5b-
reductase activity[6] occupies the same position in both the
IRIS (Tyr178) and progesterone 5b-reductase structures, as
do the NADP cofactors and the Lys146 (Lys147 in progester-
one 5b-reductase) residues, which have been proposed to be
involved in progesterone 5b-reductase catalysis. There are,
therefore, great similarities between the IRIS and progester-
one 5b-reductase structures, with the Tyr Oz being in close
T
erpenes and related isoprenoids are the largest class of
small-molecule natural products on earth, and the most
abundant by mass.[1] Most are made by enzymes that are
represented by a relatively small number of folds,[2] primarily
head-to-tail and head-to-head trans-prenyltransferases, cis-
prenyltransferases, and a range of cyclases with one, two, or
three domains. In most cases, the reactions proceed via the
formation of carbocationic transition states/reactive inter-
mediates. However, in recent work, an alternative route to the
formation of terpenes—one involving reductive cyclization—
was reported for iridoid biosynthesis.[3] Iridoids are mono-
terpenes that are produced from (C10) 10-oxogeranial (1) by
NAD(P)H-dependent reduction, followed by a cyclization
step that involves either a Diels–Alder cycloaddition or
a Michael addition.[3a,4] There are no X-ray structures
available for any iridoid synthases (IRISs), with or without
[*] Y. Hu,[+] Prof. W. Liu,[+] Prof. Y. Zheng, Prof. C.-C. Chen, Z. Xu, M. Liu,
X. Han, J. Gao, Prof. R.-T. Guo
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences, Tianjin 300308 (China)
E-mail: guo_rt@tib.cas.cn
Dr. S. R. Malwal, Dr. X. Feng, Prof. E. Oldfield
Department of Chemistry, University of Illinois
Urbana, IL 61801 (USA)
E-mail: eo@chad.scs.uiuc.edu
Dr. T.-P. Ko
Institute of Biological Chemistry, Academia Sinica
Taipei 11529 (Taiwan)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201508310.
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S5. These structures do not, how-
ever, give any new clues to the
mechanisms of catalysis.
We thus soaked 10-oxogeranial
into crystals of IRIS co-crystallized
with NAD+ (to prevent 10-oxogera-
nial substrate reduction) and
obtained an IRIS + NAD+ + 10-
oxogeranial complex structure
(PDB ID: 5DBI). Attempts to
obtain structures with nepetalactol
or progesterone (+ /ÀNADPH)
were not successful. The bound
NAD+ and 10-oxogeranial elec-
tron-density map is shown in Fig-
ure 2A, and the active-site region,
together with distances of interest,
is shown in Figure 2B. This struc-
ture is essential for a detailed anal-
ysis of the IRIS (and progesterone
reductase) mechanism of action. As
can be seen in Figure 2 (and the
ligand interaction plots in Figur-
es S4,S5), the NAD+ (NADH,
when reduced) nicotinamide, 10-
oxogeranial, and Tyr178 OzH
groups are perfectly aligned for
a 1,4-hydride addition. The pro-4(S)
proton from NADH attacks the
Scheme 1. A) Vincristine biosynthesis from isopentenyl diphosphate and dimethylallyl diphosphate.
B) Structures of other compounds of interest.
=
C2 C3 double bond from “above”
(in Figure 2B) to generate the
trans-enol, with the Tyr178 OzH
providing the enolate hydrogen
atom. This reaction is shown sche-
matically in Figure 3A. The Tyr178
Oz is only 2.5 Š from the 10-
oxogeranial O1 in the crystal struc-
ture, suggesting that there could be
a hydrogen bond present, even
prior to reduction. Upon hydride
attack, the tyrosinate formed can be
stabilized through interaction with
the ribose O2(H) (2.8 Š away in the
X-ray structure, at least prior to
reduction). The enol that forms
upon hydride attack has a 3(S)
center, as found in nepetalactol.
However, the product of this reac-
tion is actually S-10-oxocitronellal
(9), a known[4] shunt-reaction prod-
uct observed experimentally, not
nepetalactol, as shown in the Fig-
ure 3A. In order for nepetalactol to
be formed, there needs to be a 1808
rotation about C1–C2 to form the
cisoid isomer of 1, which on reduc-
Figure 1. Structure of C. roseus IRIS+NADPH and a comparison with D. lanata progesterone 5b-
reductase. A) stereo view of IRIS in complex with NADPH (PDB ID: 5DBF). B) Superimposition of
IRIS+NADPH structure (cyan) and progesterone 5b-reductase (PDB ID: 2V6G[6]) in complex with
NADP+ (yellow).
proximity to the ribose O2 (ca. 2.8 Š) in both systems.
Additional protein–ligand contacts are shown in Figures S2–
tion forms the cis-enol (Figure 3B). Rotation about C4–C5 is
also required (Figure 2B and the video S1 in the Supporting
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Figure 2. The active-site region of the C. roseus iridoid synthase from the IRIS+NAD+ +10-oxogeranial structure (PDB ID: 5DBI). A) Electron-
density map of bound NAD+ and substrate 10-oxogeranial (2FoÀFc map contoured at 1 sigma). B) Interaction of active-site residues K146 and
Y178 with NAD+ and 10-oxogeranial substrates. The C4–C5 rotation to enable cyclization is indicated. Dashed lines indicate distances of interest.
C) Progesterone modeled into the IRIS active site.
+
However, the lysine e-NH3 group is approximately
5.3 Š from the Tyr178 Oz (Figure 2B), so it seems
unlikely that this lysine plays an essential role in
reprotonating Tyr178 after 10-oxogeranial reduction.
The backbone N in Lys146 is, however, only 2.85 Š
from the 10-oxogeranial O1, suggesting a hydrogen-
bonding interaction, but this would probably be seen
with most amino acids in this position.
Based on the structural data, it appears that the
transoid form simply binds more strongly, and that is
why it is seen crystallographically. However, it is
reduced relatively slowly (in solution), which is why
S-10-oxocitronellal (9) is a minor product. The cisoid
form binds more weakly (and we do not see it), but it
reacts more rapidly (in solution), which is why, under
reducing conditions, nepetalactol is the major prod-
uct. This is, of course, a post hoc analysis, since the X-
ray structure is of the transoid form H-bonded to the
(essential) Y178 Cz-OH.
Figure 3. Proposed catalytic mechanism of iridoid synthase and progesterone
The similarities between the transoid enone frag-
reductase based on the IRIS+ NAD+ +10-oxogeranial structure (PDB ID: 5DBI).
ment in 10-oxogeranial and the same transoid moiety
A) transoid 1 gets reduced to S-10-oxocitronellal (9). B) Rotation about C1–C2
enables cis-enol formation, and with C4–C5 rotation, nepetalactol formation.
C) Progesterone reacts as in (A).
in progesterone suggested that it should be possible
to obtain a good model for progesterone binding
simply by aligning the respective enone oxygen atoms
and C3 (10-oxogeranial) and C5 (progesterone)
Information), and then cyclization and lactol formation can
proceed.
carbon atoms. This appears to be the case, since as shown in
Figure 2C, there is a very good overlap of the entire
progesterone molecule with 10-oxogeranial, an observation
that is expected to facilitate future work on progesterone (and
other enone) reductase reactions of interest in asymmetric
synthesis.
These results are also of interest in the context of the
catalytic mechanism of the homologous progesterone 5b-
reductase in plants such as D. lanata. The reduction of
progesterone to 5b-pregnane-3,20-dione (10) by progesterone
5b-reductase is expected to be similar to the proposed
reduction of the transoid form of 1 (Figure 3A), with hydride
attack on the b-face at C5 (Figure 3C). When compared with
the previous structures and mechanistic studies of progester-
one 5b-reductase,[6] it can be seen that in IRIS, the 10-
oxogeranial is immediately adjacent to Tyr178, which pro-
vides one of the protons required for catalysis, rather than
occupying a more distal location, as proposed for progester-
one 5b-reductase based on computational modeling. Tyr178,
however, is not the only residue that has been proposed to be
involved in progesterone 5b-reductase. Specifically, Lys147
was suggested to play a role[6] as a proton source. The
corresponding residue in IRIS is Lys146, which together with
Tyr178, is very well aligned (a 0.171 Š rmsd) between D.
lanata progesterone 5b-reductase and the C. roseus IRIS.
Next, we sought to determine whether there were any
other residues that might have catalytic activity in both the
IRIS and progesterone 5b-reductase reactions by using
a SCORECONS analysis[7] of a JPRED3 alignment[8] of C.
roseus iridoid synthase. SCORECONS is a computer program
that predicts the “essential” nature of a residue for catalysis,
based on its occurrence (and physicochemical similarities
between different residues) in a set of aligned sequences. A
score of 1000 means really essential, 0, not essential. A list of
the top 20 most conserved residues is shown in Table S2. The
most conserved residues are Gly residues involved in Ross-
man fold formation. Many of the other conserved residues are
hydrophobic, and all conserved residues cluster in the N-
terminal domain. Of the polar residues that might be involved
in catalysis, Arg200 and Glu182 actually form a salt bridge
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(2.9 Š Oe2/NH2) located approximately 10 Š from the
substrate C1. Asp84 forms a salt bridge with the adenine
N6. Tyr178 thus appears to be essential for catalysis in both
proteins, since it is highly conserved and is positioned
immediately adjacent to the 10-oxogeranial substrate.
To help test the proposed roles of this and other residues
in or near the (putative) catalytic center, we produced seven
IRIS single-point mutants: K146A, F149M, Y178F, Y178A,
F342A, A346I, and S349F. Enzyme activity results are shown
in Figure 4A and a “heat map” of the results (red = essential;
with large enones (e.g., progesterone) they again act as
“gatekeepers”, this time impeding access to the active site.
Phe!Ala mutations of several PRISEs[3e] favor progesterone
reduction but disfavor small enone reduction. This is con-
sistent with what we see with IRIS, in which the F342A
mutant is inactive for 10-oxogeranial reduction.
In summary, the iridoid synthase that produces nepeta-
lactol in C. roseus has a structure that is very similar to that
found for progesterone 5b-reductase from D. lanata. This
iridoid synthase can produce two different products: reduc-
tion of the transoid substrate seen crystallographically results
in formation of the side-product S-10-oxocitronellal, whereas
reduction of the cisoid form, together with a C4–C5 rotation,
enables cyclization and formation of the major product,
nepetalactol. The Tyr178 CzOH group provides the proton
required for enol formation, the Lys146 HN can form H-
bonding interactions with the substrate O1, and the tyrosinate
produced in the first half reaction can be stabilized by H-
bonding to the ribose O2H. There are no other acidic groups
close by that appear to be involved in catalysis, although
Phe342 is essential for 10-oxogeranial reduction, which is
consistent with the progesterone-5b-reductase mutagenesis
results with small enone substrates.[3b] Overall, these results
are of broad general interest since they provide new insight
into the structures and catalytic mechanisms of iridoid
synthases/progesterone 5b-reductases, enzymes that are
involved in the biosynthesis of important cancer and heart
drugs [indole terpene alkaloids such as vincristine (2) and
cardiac glycosides such as digitoxin (4)], in addition to
providing structural models for the future development of
asymmetric catalysts.
Acknowledgements
We thank the National Synchrotron Radiation Research
Center of Taiwan for beam time allocation and data collection
assistance. This work was supported by National High
Technology Research and Development Program of China
(2012AA022209), the National Natural Science Foundation
of China (31200053, 31400678 and 31300615), the United
States Public Health Service (NIH grants CA158191,
GM065307), a Harriett A. Harlin Professorship and the
University of Illinois Foundation/Oldfield Research Fund.
Figure 4. Mutagenesis results for C. roseus iridoid synthases. A) Activ-
ities of wild-type and mutant IRIS proteins for 10-oxogeranial reduc-
tion. B) “Heat map” showing the effects of mutations on catalytic
activity: green=no effect; yellow=significant effect; red=major effect
(no activity, within experimental error).
green = non-essential, yellow = desirable) is shown in Fig-
ure 4B. These results confirm the essential nature of Tyr178
for activity. Phe342 is also essential, and this result is of
interest since it supports the role of the equivalent Phe
residue in progesterone reductase in small-enol reductase
activity,[3b] while Phe149 is not essential, which is consistent
with its non-conserved nature. Lys146 has reduced activity,
but no obvious role for this residue is suggested from the X-
ray structure. It might be involved in the second half reaction,
but it is clearly not absolutely essential (as also noted in
progesterone reductase by Petersen et al).[3e] Ala346 and
Ser349, which are close to the substrate, are also not essential
for 10-oxogeranial reduction, which is again consistent their
non-conserved nature. Based on Phe mutagenesis and MD
simulations, Petersen et al. propose that several Phe residues
(that are close to the substrate in our structure) play a dual
role. In IRIS or IRIS-like reduction reactions involving small
1,4-enones, they help keep ligands inside the protein, while
Keywords: biosynthesis · enzyme mechanisms ·
monoterpenes · natural products · X-ray crystallography
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Received: September 5, 2015
Revised: October 8, 2015
Published online: November 13, 2015
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