Terpene cyclization mechanism
SS Shinde et al
4
[2H]-1. During the MS analysis, we found that fragment F257 consisted rearrangement of 1. The resultant 12-membered ring cation 11+ then
of two isomeric fragments, which could be separated during the undergoes a C2–C10 cyclization to afford 5-5-9 tricyclic cation 12+.
fragmentation of specifically labeled [2H]-1 (Figures 1d,e and
Subsequent 1, 2-hydride shift and C2–C6 ring closure followed by
Supplementary Figure S3). These fragments may provide further
confirmation of the labeling pattern upon detailed HR-MS and MS/
MS analyses. However, these experiments were not performed since
sufficient data were obtained from other fragment ions.
C6-deprotonation furnish 1.
Sesterterpene mangicol A, derived from a marine fungus, possesses
a unique tetracyclic scaffold, which is closely related to 1. Its
biosynthetic pathway resembles that of 1 and was proposed by feeding
experiments of various 13C-labeled acetates.18 A branch point
between the metabolic pathways of 1 and mangicol is the cyclization
of 11+/11a+. Instead of the C2–C10 cyclization in the pathway of 1, an
alternative C6–C10 cyclization leads to the core structure of mangicol
A via a key 5-9-5 tricyclic intermediate (Supplementary Scheme S2).
In 2015, we proposed that the amino acid sequence of the terpene
cyclase domain of bifunctional terpene synthases might reflect the
first cyclization mode (Figure 3).9 A phylogenetic analysis showed that
PaPS could be classified into clade B, which catalyzes the formation of
a 5–11 ring system via a C1-III-IV cyclization (type B cyclization).
The elucidated cyclization mechanism of PaPS is consistent with our
proposal. Furthermore, this has been extended to two more clade
B-bifunctional terpene synthases, that is, astellifadiene synthase and
stellatatriene synthase, which have recently been identified.19,20
In this study, we developed an efficient method to synthesize site-
specifically isotope-labeled oligoprenyl diphosphate precursors, such as
DMAPP, GPP, FPP and GGPP, starting from four chemically
synthesized [2H]-IPPs by enzymatic transformation. By using these
precursors, we demonstrated that the MS-based analysis of site-
specifically isotope-labeled terpenes provides sufficient information
to elucidate the cyclization mechanism catalyzed by PaPS. During this
study, we proposed a general strategy for the assignment of individual
fragment ions: (1) information on the location of each isoprene unit
(outline of fragmentation) could be obtained by analysis of the
samples from the [C2H3]-labeled precursors; (2) detailed MS analysis
of terpenes from isoprene unit-specifically labeled precursors provided
useful information to assign the structure of the eliminated fragments;
(3) fragmentation process of terpene could be proposed on the basis of
chemical structure of cyclic terpene and labeling pattern of the
eliminated fragments. In addition, overlapped ions with the same
molecular m.u. could be separated by using a [C2H3]-labeled
precursor and further analysis of 3 m.u.-shifted peaks provided
additional information.
In the cyclization with various terpene synthases, most of methyl
groups on the isoprene unit of a prenyl diphosphate remain intact in
the cyclization product. Thus, we speculated that, in the MS analysis of
1, methyl groups could be useful fragmentation tracers to identify
fragments derived from the specific isoprene units I–VI in GGPP.
To obtain information on the origin of the methyl groups in the
fragments, we analyzed the MS spectra from the enzymatic reactions
with multiply and specifically [C2H3]-labeled GGPP. The results
showed that the labels in the isoprene units I, II, III, IV were located
at C20, C19, C18 and C16/C17, respectively (Figure 1f and
Supplementary Figure S3).
Considering the proposed structures of the key fragments,
we initially focused on the formation of an A-ring that could most
likely originate from isoprene units III and IV based on the structure
of 1. Four fragments, F243, F216, F202 and F105, were useful to examine
this mechanism (Figure 2). Mass shifts between non-labeled and
labeled-1 enabled us to speculate the labeling pattern of 1. Isobutene
unit D (M-F216) from IV-[1′-CD2]- and IV-[5′-CD3]-GGPP retained
the labels (Figures 2a and b), whereas C5 unit A (M-F202) possessed
additional labels from III-[4′-CD2]-GGPP (Figure 3c). The neutral
C3 unit C (F202-F160-F145-F105) from IV-[2′-CD] and III-[5′-CD3]-
GGPP (Figures 2d and e) and ethylene unit E (M-F243) from
IV-[1′-CD2]- and III-[4′-CD2]-GGPP (Figures 2a and c) were
eliminated with the labels. Consequently, the origin of all the hydrogen
atoms on the A-ring was confirmed, as shown in Figure 2.
Next, we examined the formation of a C/D-ring that originated
from isoprene units I and II. Propene unit B (F202-F160) possessed
the labels from I-[2′-CD], I-[4′-CD2] and I-[5′-CD3]-GGPP
(Figures 2f–h). By contract, no deuterium label (M272) from
II-[2′-CD]-GGPP was observed (Figure 2i), while other labels from
II-[1′-CD2], II-[4′-CD2] and II-[5′-CD3]-GGPP were all retained
(Supplementary Figure S3). Most of the remaining labels of units
I-III were found in F160. An exceptional labeling pattern was observed
in the analysis of 1 from III-[2′-CD] and I-[1′-CD2]-GGPP. Although
1 retained the labels, the expected MS shift of F202 was not observed
in these cases. We speculated that an unexpected hydrogen scrambling
occurred at these positions. However, this does not influence our
proposal for the cyclization mechanism.
For the A-ring formation, labels from unit IV in GGPP located at
C13 and C14-C15(C17)-C16 in 1, indicated that a C13–C14 bond
cleavage occurred during the cyclization. Isoprene unit III C9–C10 and
C11(C18)-C12 in 1 was separated by the insertion of C14 between
C10 and C11. The labeling pattern revealed that two rounds of
1,2-C-shifts are involved in the A-ring construction. In the D-ring
construction, the labels from I-[2′-CD], I-[4′-CD2] and I-[5′-CD3]-
GGPP were located on the D-ring at C3-C4-C20, respectively,
suggesting that the C2-olefinic proton of unit I in GGPP was shifted
on C3 in 1. The loss of the label from II-[2′-CD]-GGPP in 1 suggested
that the final quenching of the C7-carbocation occurred during
the deprotonation at C6 to afford 1. Overall, the cyclization mechan-
ism of PaPS is summarized in Scheme 2. The cyclization begins with
the elimination of the pyrophosphate group of GGPP followed by
a cyclization to afford dolabelladien-15-yl cation (10+). Two sequential
This highly sensitive method can be applied to a number of
structurally complex terpenes. It was pointed out that a careful
interpretation is required to perform the analysis due to the
non-specific hydrogen migrations during the fragmentation processes
and the kinetic isotope effects.21 These may be overcome by further
confirmation of the labeling pattern by detailed HR-MS and MS/MS
analyses, which were not used in this study. Recently, a systematic
investigation into the EI-MS fragmentation mechanism of sesquiter-
penes using all possible 13C-labeled isotopomers of FPP was also
reported.21,22 Thus, several methods for investigating the cyclization
mechanism of terpene synthases are now available. These mechanisms
will contribute to the rational engineering of terpene synthases for the
generation of diverse terpene skeletons.
METHODS
General
Oligonucleotides for polymerase chain reaction (PCR) were purchased from
Hokkaido System Science Co., Ltd. Sequence analysis of the PCR fragment was
performed by an automatic DNA sequencer (Applied Biosystems, Foster, CA,
USA, ABI PRISM 310 Genetic Analyzer). Cell disruption was dealt with a
1, 2-alkyl shifts are the key processes accounting for the carbon ultrasonic disrupter UR-200P (TOMY SEIKO, Tokyo, Japan). Analysis of the
The Journal of Antibiotics
Shinde, Sandip S
