Deciphering the Biosynthetic Mechanism of Pelletierine in Lycopodium Alkaloid Biosynthesis

Article abstract of DOI:10.1021/acs.orglett.0c03339

Pelletierine, a proposed building block of Lycopodium alkaloids (LAs), was demonstrated to be synthesized via the non-enzymatic Mannich-like condensation of Δ1-piperideine and 3-oxoglutaric acid produced by two new type III PKSs (HsPKS4 and PcPKS1) characterized from Huperzia serrata and Phlegmariurus cryptomerianus, respectively. The findings provide new insights for further understanding the biosynthesis of LAs such as huperzine A.

Full text of DOI:10.1021/acs.orglett.0c03339

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Letter
Deciphering the Biosynthetic Mechanism of Pelletierine in
Lycopodium Alkaloid Biosynthesis
Juan Wang,∥ Ze-Kun Zhang,∥ Fang-Fang Jiang, Bo-Wen Qi, Ning Ding, Saw Yu Yu Hnin, Xiao Liu,
Jun Li, Xiao-hui Wang, Peng-Fei Tu,* Ikuro Abe,* Hiroyuki Morita,* and She-Po Shi*
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ABSTRACT: Pelletierine, a proposed building block of Lycopo-
dium alkaloids (LAs), was demonstrated to be synthesized via the
non-enzymatic Mannich-like condensation of Δ1-piperideine and
3-oxoglutaric acid produced by two new type III PKSs (HsPKS4
and PcPKS1) characterized from Huperzia serrata and Phlegmar-
iurus cryptomerianus, respectively. The findings provide new
insights for further understanding the biosynthesis of LAs such
as huperzine A.
Lycopodium alkaloids (LAs) represent an important class of
nitrogen-containing heterocyclic metabolites with remarkably
diverse and stereochemically complex structures, exhibiting
significant biological functions such as acetylcholine esterase
(AChE) inhibitory, cytotoxic, and neuroprotective activities.1
Scheme 1. Proposed Biosynthetic Pathway of Huperzine A
and Related Lycopodium Alkaloids
Since the pioneering isolation of lycopodine from Lycopodium
2
̈
complanatum by Bodeker in 1881, more than 400 LAs have
been obtained from the families of Lycopodiaceae and
Huperziaceae.1,3 Among the hitherto reported LAs, huperzine
A is a typical representative with efficient, reversible, and highly
selective inhibitory activities against AchE and is therefore
being used as a promising therapeutic agent for the treatment
of Alzheimer’s disease.4 Given their unique and intriguing
structures, biogenesis, and wide-ranging biological activities,
LAs have attracted immense interest worldwide; in particular,
chemical strategies for the construction of LA scaffolds have
been extensively reported.5 In contrast, the biosynthetic
mechanism of LAs remains elusive due to major difficulties
in the cultivation and in vitro propagation of LA-producing
plants. Despite the lack of direct biochemical evidence, a
plausible biogenetic pathway for LAs has been proposed on the
basis of feeding experiments (Scheme 1) as follows. The
biosynthesis of LAs is initiated by the decarboxylation of lysine
to produce cadaverine, with the subsequent oxidative
deamination of cadaverine to form 5-aminopentanal, which
could be immediately converted to Δ1-piperideine by
spontaneous cyclization. The Δ1-piperideine then couples
with 3-oxoglutaric acid (or its CoA thioester) from the
condensation of two malonyl-CoAs to generate 4-(2-piperidyl)
acetoacetate (4PAA) or 4PAA-CoA, followed by the
decarboxylation of 4PAA to produce pelletierine. Thereafter,
the 4PAA condenses with pelletierine to form the universal
precursor phlegmarine that undergoes cyclization, multistep
oxidation, and ring cleavage reactions to produce structurally
diverse LAs.6 Following the biosynthetic pathway mentioned
above, lysine decarboxylase (LDC) and copper amine oxidase
(CAO) have been characterized from Huperzia serrata and
demonstrated to play a crucial role in the formation of 5-
Received: October 6, 2020
https://dx.doi.org/10.1021/acs.orglett.0c03339
© XXXX American Chemical Society
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A

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Letter
aminopentanal from lysine (Scheme 1).7 However, other
enzymes involved remain completely unknown.
In this study, the biosynthetic mechanism of pelletierine, a
proposed intermediate in LA biosynthesis, was deciphered with
the characterization of two new 3-oxoglutaric acid-producing
type III polyketide synthases (PKSs), HsPKS4 from H. serrata
and PcPKS1 from Phlegmariurus cryptomerianus.
oxo-4-(piperidin-2-yl)butanoic acid (P01) and pelletierine
(P02), respectively. To determine the structures of these two
products, a large-scale reaction was performed. However,
limited by the very weak UV absorption of P01 and P02,
purification of these two products by HPLC is very
challenging. Therefore, the reaction solution, after being
incubated at 37 °C for 10 h, was adjusted to pH 9.0 with
sodium hydroxide. Fluoro-2,4-dinitrobenzene (FDNB) was
then added to the reaction mixture as a derivatization reagent
that could readily convert the secondary amine-containing
products to their corresponding DNB derivatives (Figure S5).
Further separation of the reaction mixture using HPLC
expectedly yielded DNB-P02, but DNB-P01 could not be
purified due to its propensity to be converted into DNB-P02
via spontaneous decarboxylation. On the basis of the
HRESIMS, UV, and NMR data, the structure of DNB-P02
was unambiguously assigned as DNB-pelletierine (Figures S6
and S7). The HRESIMS spectrum of DNB-P02 showed the
presence of a [M + H]+ ion peak at m/z 308.1245, consistent
with the empirical molecular formula C14H18N3O5 (calcd m/z
308.1241). In the NMR spectra of DNB-P02, the characteristic
signals due to the DNB moiety could be observed. In addition,
resonances contributing to a pelletierine moiety could also be
found at δH 1.50−1.80 (5H, m), 1.93 (1H, m), 2.15 (3H, s),
2.69 (1H, dd, J = 17.5, 6.0 Hz), 3.05 (1H, dd, J = 17.5, 6.5
Hz), 2.80 (1H, br d, J = 13.0 Hz), 3.25 (1H, m), and 4.61 (1H,
m) and δC 205.8, 53.2, 47.4, 43.4, 30.8, 29.1, 25.8, and 18.8.
However, the near-zero optical rotation value suggested that
DNB-P02 might be a racemate, which could be further
confirmed by the successful separation of DNB-P02 on a chiral
column to obtain two optically pure compounds, (+)-DNB-
P02 and (−)-DNB-P02, sharing almost equal optical values
Using a transcriptomic sequence data mining strategy, one
putative type III PKS unigene (Hspks4), in addition to the
known Hspks1 and Hspks3,8 and two (Pcpks1 and -2) were
obtained from LA-producing plants H. serrata and P.
cryptomerianus, respectively. The three new PKSs (HsPKS4,
PcPKS1, and PcPKS2) show 50−65% amino acid identity with
other plant PKSs, maintaining a Cys-His-Asn catalytic triad
commonly present in all known type III PKSs (Figure S1).9
Phylogenetic analysis revealed that they were all grouped into
nonchalcone synthases (Figure S2). Three recombinant
proteins with molecular weights of approximately 42−46
kDa were successfully obtained by heterologous expression in
Escherichia coli and affinity chromatography purification
(Figure S3).
All three recombinant proteins could not accept most acryl-
CoAs, such as p-coumaroyl-, coumaryl-, feruloyl-, benzoyl-, and
hexanoyl-CoA, as a starter to perform iterative decarboxylative
condensation reactions with malonyl-CoA. However, when
HsPKS4 was incubated with Δ1-piperideine and malonyl-CoA
for 10 h, the reaction generated a major product P01 with the
molecular formula C9H15NO3, which is determined by the
presence of a [M + H]+ ion peak at m/z 186.1113 (calcd for
C9H16NO3, m/z 186.1125) in its HRESIMS spectrum (Figure
1A,B). In addition, P02, with the molecular formula C8H15NO
supposedly being the decarboxylation product of P01, could
also be detected from the reaction mixture (Figure 1A,C). In
the MS2 spectra of P01 and P02 (Figure S4), the observation
of the typical fragment ion at m/z 84 contributing to a
piperidinyl moiety suggested that P01 and P02 might be 3-
25
([α]D = +306, and −300, respectively) (Figure 2). On the
Figure 2. Derivatization of the enzymatic products with fluoro-2,4-
dinitrobenzene (FDNB) and HPLC chromatogram for the chiral
separation of racemic ( )-DNB-P02 on a CHIRALPAK IA (5 μm,
4.6 mm × 150 mm) column.
basis of the structure of DNB-P02, the structures of P01 and
P02 could be determined as ( )-3-oxo-4-(piperidin-2-yl)-
butanoic acid and ( )-pelletierine, respectively. Notably, trace
amounts of P03 featuring two piperidinyl moieties could also
be detected from the reaction mixture (Figure 1A), and its
structure was presumably identified as anaferine on the basis of
the observation of the fragment of pelletierine (m/z 142.1234)
produced by losing a piperidinyl moiety (Figure 1D).
Here, the nonstereospecific incorporation of the C3 side
chain in pelletierine seems quite different from a very similar
reaction catalyzed by carboxymethylproline synthases (CarB)
in which a C2 side chain from malonyl-CoA can be
stereospecifically incorporated into L-pyrroline-5-carboxylate.10
Accordingly, we proposed that the C3 side chain in P01 and
Figure 1. (A) Extracted ion chromatograms for enzymatic products
P01−P03 produced by HsPKS4 from malonyl-CoA and Δ1-
piperideine. MS spectra and structures of (B) P01, (C) P02, and
(D) P03.
B
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P02 might be integrated with Δ1-piperideine via a non-
enzymatic Mannich-like condensation in a manner similar to
the previously reported formation of 4-(1-methyl-2-pyrroli-
dinyl)-3-oxobutanoic acid in the biosynthesis of tropane
alkaloids by an unusual type III PKS named pyrrolidine ketide
synthase (PYKS).11 Therefore, Δ1-piperideine and 3-oxo-
glutaric acid were incubated together with and without
HsPKS4. As expected, all three products (P01−P03) could
be produced in the absence of an enzyme (Figure S8B), and
the reaction velocity of the two reactions seems the same
(Figure S9), which demonstrated that the C3 side chain was
incorporated via a non-enzymatic reaction. Then, malonyl-
CoA, in the absence of Δ1-piperideine, was incubated with
HsPKS4, and the reaction expectedly generated a product
(P04) with a molecular formula of C5H6O5 assigned by the
presence of a [M + K]+ ion peak at m/z 184.9850 (calcd for
C5H6O5K, m/z 184.9847) in its HRESIMS spectrum (Figure
3). P04 was unequivocally identified as 3-oxoglutaric acid by
AaPYKS with Thr143, Ser143, and Thr152, respectively, do
not have any 3-oxoglutaric acid-forming activity unlike that of
HsPKS4 and PcPKS1. The evidence mentioned above
suggested that the two PKSs (HsPKS4 and PcPKS1) reported
here might use a catalytic mechanism similar to that of
AaPYKS to generate 3-oxoglutaric acid. Supposedly, both
HsPKS4 and PcPKS1 first catalyzed the single-round
condensation of two molecules of malonyl-CoA to produce
3-oxoglutaric acid, which could undergo non-enzymatic
Mannich-like condensation with Δ1-piperideine to form
racemic 3-oxo-4-(piperidin-2-yl)butanoic acid (P01) and
then with decarboxylation of P01 to generate racemic
pelletierine (P02). In addition, P01 is non-enzymatically
condensed with another molecule of Δ1-piperideine to
generate P03 (Scheme 2). It should be noted that for the C3
Scheme 2. Proposed Reaction Mechanism for the
Formation of Pelletierine from Malonyl-CoA and Δ1-
Piperideine
Figure 3. Extracted ion chromatograms at [M + K]+ = 184 of product
P04 in enzymatic reactions by HsPKS1, HsPKS3−4, and PcPKS1−2
using malonyl-CoA as the solo substrate.
side chain in pelletierine and its biogenetically downstream
products, phlegmarine-type alkaloids isolated from natural
plants are coincidentally incorporated in nonstereospecific
modes,12 which strongly supports that the condensation of Δ1-
piperideine and 3-oxoglutaric acid in the biosynthesis of LAs is
a non-enzymatic reaction. Although the biosynthetic mecha-
nism of pelletierine reported here is similar to the biosynthesis
of tropane alkaloids recently reported by Huang,11b this is the
first study to decipher the biosynthetic mechanism of
pelletierine with direct biochemical evidence. The decarbox-
ylative coupling of P01 and P02 produces phlegmarine, which
is the common biosynthetic precursor of all LAs.
In summary, this study successfully characterized two new 3-
oxoglutaric acid-producing type III PKSs from LA-producing
plants and subsequently demonstrated, for the first time, the
biosynthetic mechanism of pelletierine, which is supposedly a
universal building block of all LAs. The findings provide new
insights into further understanding the biosynthesis of LAs,
which include huperzine A, a promising anti-Alzheimer’s
disease drug candidate.
comparing its retention time on the HPLC chart and MS data
with those of the authentic compound. In contrast, although
several approaches have been attempted, 3-oxoglutaric acid
CoA ester could not be detected in the reaction solution or the
directly lyophilized reaction mixture, suggesting that HsPKS4
might catalyze 3-oxoglutaric acid formation.
Remarkably, PcPKS1 from another LA-producing plant, P.
cryptomerianus, could also catalyze the formation of 3-
oxoglutaric acid (P04), indicating the same catalytic functions
as those of HsPKS4. Homology modeling studies of HsPKS4
(Figure S10) predicted that HsPKS4 posed a downward
expanded active site cavity compared with that of AaPYKS
with unique 3-oxoglutaric acid-forming activity,11b due to the
substitutions of Leu256, Leu258, and Phe267 lining the active
site cavity of AaPYKS with Val249, Gly251, and Tyr260 in
HsPKS4, respectively. However, the Cys-His-Asn catalytic
triad and key residues Arg134, Met139, Phe217, and Ser340,
which play a crucial role in determining the unqiue 3-
oxoglutaric acid-forming activity of AsPYKS, were conserved in
HsPKS4 with the same residues in the same location and
orientation, very similar to what occurs in AaPYKS.
Furthermore, no other residues that could act as the acid−
base catalyst appeared in the HsPKS4 homology model. A
homology model of PcPKS1 (Figure S10) also showed the
same active site architecture as that of HsPKS4 due to the
active site cavity being composed of the same residues as those
of HsPKS4. In contrast, the other three enzymes (HsPKS1,
HsPKS3, and PcPKS2) with the replacement of Arg134 in
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c03339.
Experimental section (general experimental procedures,
cDNA cloning, expression of cDNA, enzyme purifica-
tion, and enzyme reaction), phylogenetic analysis,
alignment of amino acid sequence, full spectroscopic
data (NMR and HRESIMS) for DNB-P02, and LC-MS
data for enzymatic reactions (PDF)
C
https://dx.doi.org/10.1021/acs.orglett.0c03339
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AUTHOR INFORMATION
Corresponding Authors
■
She-Po Shi − Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, China; orcid.org/0000-0003-3252-3108;
Email: shishepo@163.com
Hiroyuki Morita − Institute of Natural Medicine, University of
Toyama, Toyama 930-0194, Japan; Email: hmorita@inm.u-
toyama.ac.jp
Ikuro Abe − Graduate School of Pharmaceutical Sciences, The
University of Tokyo, Tokyo 113-0033, Japan; orcid.org/
0000-0002-3640-888X; Email: abei@mol.f.u.-tokyo.ac.jp
Peng-Fei Tu − Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, China; orcid.org/0000-0003-3553-1840;
Email: pengfeitu@163.com
̈
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Authors
Juan Wang − Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, China
Ze-Kun Zhang − Modern Research Center for Traditional
Chinese Medicine, Beijing University of Chinese Medicine,
Beijing 100029, China
Fang-Fang Jiang − Modern Research Center for Traditional
Chinese Medicine, Beijing University of Chinese Medicine,
Beijing 100029, China
Bo-Wen Qi − Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, China
Ning Ding − Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, China
Saw Yu Yu Hnin − Institute of Natural Medicine, University of
Toyama, Toyama 930-0194, Japan
Xiao Liu − Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, China
Jun Li − Modern Research Center for Traditional Chinese
Medicine, Beijing University of Chinese Medicine, Beijing
100029, China; orcid.org/0000-0001-8243-5267
Xiao-hui Wang − Modern Research Center for Traditional
Chinese Medicine, Beijing University of Chinese Medicine,
Beijing 100029, China
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Complete contact information is available at:
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Author Contributions
∥J.W. and Z.-K.Z. contributed equally to this work.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (81773842 and 81573312) and
the Drug Innovation Major Project (2018ZX09711001-006).
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