Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases

Article abstract of DOI:10.1021/acschembio.5b00539

Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis.

Full text of DOI:10.1021/acschembio.5b00539

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Articles
pubs.acs.org/acschemicalbiology
Mechanism-Based Post-Translational Modification and Inactivation
in Terpene Synthases
Roland D. Kersten, Jolene K. Diedrich, John R. Yates, III,‡,§ and Joseph P. Noel*,†
†
‡,§
†Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology & Proteomics, The Salk Institute for Biological
Studies, La Jolla, California 92037, United States
‡
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, United States
§
Vincent J. Coates Mass Spectrometry Center, The Salk Institute of Biological Studies, La Jolla, California 92037, United States
*
S Supporting Information
ABSTRACT: Terpenes are ubiquitous natural chemicals with
diverse biological functions spanning all three domains of life.
In specialized metabolism, the active sites of terpene synthases
(
TPSs) evolve in shape and reactivity to direct the biosynthesis
of a myriad of chemotypes for organismal fitness. As most
terpene biosynthesis mechanistically involves highly reactive
carbocationic intermediates, the protein surfaces catalyzing
these cascade reactions possess reactive regions possibly prone
to premature carbocation capture and potentially enzyme
inactivation. Here, we show using proteomic and X-ray
crystallographic analyses that cationic intermediates undergo
capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation
increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and
alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation
then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation
represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis.
iverse terpene-based natural products are widespread
across all three domains of life. In the specialized
TPSs typically catalyze cationic mechanisms involving
14
D
multiple highly reactive intermediates. The catalytic cycle of
a sesquiterpene synthase begins with binding of the
diphosphate moiety of FPP to positively charged residues and
Mg2+ cofactors coordinated by two Mg -binding elements, the
metabolism of bacteria, plants, fungi, and animals, terpene
natural products are used for defense and attraction, ecological
2+
1
−4
communication, and physiologic regulation.
Furthermore,
“
DDxxD” motif and the “NSE/DTE” motif, both sitting at the
cyclic terpene structures like sterols and hopanes serve as
15
5
,6
entrance to the active site. The farnesyl chain tucks into the
hydrophobic and aromatic-rich portion of the active site
important membrane constituents in all life forms. Many
cyclic terpenes are also commercially important compounds
used as agrochemicals, pharmaceuticals, flavors, and fragran-
(
Figure 1A). Subsequently, the carbon−phosphoester bond of
7
−10
FPP breaks through ionization, and an allylic farnesyl cation
forms. The farnesyl cation then undergoes cationic reaction
cascades consisting of electrophilic additions, deprotonation−
reprotonation reactions, and hydride/alkyl rearrangements
mediated by the shape and chemistry of the active site. The
high reactivities of cationic intermediates are balanced in TPSs
by hydrophobic active sites and several aromatic active site
ces.
For terpene natural products such as 15-carbon sesquiter-
penes, their functional complexity centers on the structural
diversity of multicyclic, chiral hydrocarbon scaffolds most
commonly biosynthesized starting with the primary metabolite,
11
(
E,E)-farnesyl diphosphate (FPP) (Figure S1). Plant class I
16
TPSs consist of two α-helical domains, while bacterial and
residues that stabilize cations through π-cation interactions. In
addition, induced-fit closures of the active sites after substrate
binding and ionization by C-terminal loops limit premature
11
fungal class I TPSs contain a single α-helical catalytic domain.
While the N-terminal domains in plant class I TPSs appear to
lack catalytic activity, their C-terminal domains and the single
helical domains of bacterial and fungal TPSs contain the active
17,18
solvent quenching.
Reaction termination occurs through
cation neutralization. Three possible cation-based quenching
scenarios exist: (1) cation neutralization through proton loss
2
+
sites employing conserved Mg -binding motifs and variable
surrounding residues. Together, these motifs catalyze the
ionization of FPP and the ensuing structural diversity of cyclic
sesquiterpene scaffolds through carbocation-based mecha-
Received: July 13, 2015
Accepted: August 20, 2015
1
2,13
nisms.
©
XXXX American Chemical Society
A
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Figure 1. Structural basis of cyclic sesquiterpene biosynthesis and carbocation quenching scenarios in sesquiterpene synthases. (a) Active site model
1
9
of wild-type TEAS with bound substrate mimic 1-hydroxyfarnesyl phosphonate (colored yellow) based on Protein Data Bank entry 5EAT.
Selected active site residues are displayed and numbered. The dynamic active site J/K loop is colored magenta. Three Mg2 ions are highlighted as
gray spheres and waters as red crosses. Active site mutation sites for alkylation studies (Table 1) are colored orange. (b) Carbocation quenching
scenarios in sesquiterpene synthases. During enzyme-catalyzed sesquiterpene cyclization, cationic intermediates are neutralized by deprotonation as
exemplified by the proposed last step in 5-epi-aristolochene biosynthesis, quenching with an exogenous nucleophile such as water exemplified by
the proposed last step in patchoulol biosynthesis, or quenching with an endogenous nucleophile such as an active site residue to yield an alkylated
+
1
9
9
active site. Biosynthetic steps in TPS active sites are highlighted in gray.
1
9,20
from a neighboring carbon,
(2) quenching through
mechanistically complex, ecologically important, and commer-
cially promising enzymes are additional constraints on TPS
biosynthetic diversification.
10
nucleophilic attack by an exogenous species such as water,
and (3) nucleophilic substitution by a reactive enzyme side
chain (Figure 1B). Scenarios 1 and 2 yield sesquiterpene and
sesquiterpenol products, respectively (Figure 1B). Scenario 3,
however, lead to active site alkylations of TPSs possibly
resulting in mechanism-based inhibition by covalently bound
biosynthetic intermediates.
Mechanism-based enzyme inhibition includes inactivation of
enzymes by turnover of substrate analogues, resulting in
covalent attachment to amino acid residues or cofactors in
RESULTS
■
Self-Alkylation of Tobacco 5-epi-Aristolochene Syn-
thase (TEAS) Mutants. To characterize self-alkylation in a
sesquiterpene synthase, we chose tobacco 5-epi-aristolochene
synthase (TEAS) as a model enzyme, as X-ray crystallography
methods are well-established for facile determination of TEAS
19
X-ray crystal structures (Figure 1A). Self-alkylations by active
21
enzyme active sites. Mechanism-based inhibition has been
site changes were investigated for two groups of TEAS mutants.
The first group of mutants possessed single active site changes
via introduction of nucleophilic side chains such as cysteines or
glutamates (Table 1). The second group of mutants focused on
single active site changes causing subtle steric changes to the
active site with minimal alterations in side chain chemistry
21
extensively studied for drug discovery. Although enzymatic
self-inactivation by oxidative damage occurs in many heme-
dependent enzymes such as prostaglandin endoperoxide H
the occurrence of mechanism-based inhibition of
TPSs has received little attention. Because sesquiterpene
synthase active sites undergo changes in shape and reactivity
2
2,23
synthase,
2+
19
(
Table 1). The Mg -binding sites were not mutated to
12,13
24
2+
during TPS evolution
and upon protein engineering, self-
maintain Mg coordination essential for TPS activity. To
detect site-specific alkylation of TEAS, the reaction mixtures of
TEAS mutants or wild-type TEAS and (E,E)-FPP were
analyzed by bottom-up proteomics. The masses of nonalkylated
and alkylated tryptic peptides were calculated and identified in
the corresponding LC−MS/MS data sets. When charge states
of detected peptide mass signals (usually z = 1−3) were taken
into account, the detection of tryptic peptide mass signals
alkylations may represent significant selective pressures on TPS
2
5
evolution and engineering. In addition to mutational
modulation of the TPS itself, self-alkylation may also result
from the use of nonconventional or synthetic substrates, as
recently demonstrated by the mechanism-based inhibition of
aristolochene synthase of Penicillium roquefortii with vinyl-
26
farnesyl diphosphate.
+
Here, we investigate the occurrence, location, and chemistry
of inhibitory self-alkylations in both specific and promiscuous
sesquiterpene synthases from plants and fungi using proteomics
and X-ray crystallography. Our in vitro characterizations of self-
alkylations in sesquiterpene synthases in response to active site
mutations, substrate changes, and reaction temperatures
strongly suggest that mechanism-based alkylations during the
course of natural evolution and synthetic engineering of these
shifted by 204.188 Da (C H , i.e., m/z 204.2 for z = 1, m/z
15
24
102.1 for z = 2, and m/z 68.1 for z = 3) was indicative of
alkylated tryptic peptides. A monoalkylation mass shift of
204.188 Da was detected for all mutants except I294L on the
tryptic peptide V[442−453]K residing on the active site surface
(Table 1, Figure 2A,B, and Figures S2 and S3). In some cases,
TEAS mutants, including W273C, W273E, W273F, Y404F,
Y404C, and L407I, showed an alkylation mass shift on the
B
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C
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Figure 2. Self-alkylation analysis of TEAS active site mutant W273E with (E,E)-farnesyl diphosphate. (A) LC−MS analysis of a tryptic digest of
TEAS W273E after reaction with (E,E)-farnesyl diphosphate. Two tryptic peptides encompassing active site residues, V[442−453]K and I[515−
5
32]K, showed an alkylation mass shift (red chromatograms). (B) MS analysis of alkylated and nonalkylated tryptic peptides V[442−453]K and
I[515−532]K. For detailed MS/MS analysis, see Figure S2. (C) X-ray crystallographic analysis of alkylated TEAS W273E reveals a C1-farnesyl-γO-
aspartate ester at Asp444. (D) X-ray crystallographic analysis of nonalkylated TEAS W273E. The electron density from unbiased (i.e., simulated-
annealing omit) sigma-A-weighted 2F − F maps is shown at contour level 0.8σ with refined active site models in panels C and D. (E)
Characterization of alkylation site Asp444 by EThcD-MS/MS analysis of alkylated tryptic peptide V[442−453]K. See also Figure S5. Abbreviations:
BPC, base peak chromatogram; m(obs), observed mass; m(calc), calculated mass; ESI, electrospray ionization; Δm, mass error; EThcD, electron
transfer and high-energy collision dissociation; FTMS, Fourier transform ion cyclotron resonance mass spectrometry.
o
c
tryptic active site peptide I[515−532]K (Table 1, Figure 2B,
and Figures S2 and S3). Quite surprisingly, no alkyl mass shifts
were detected on peptides spanning the introduced cysteine or
glutamate residues as putative cation traps (W273C, W273E,
and Y404C) (Table 1 and Figure S3). V[442−453]K spans the
mutant on the V[442−453]K tryptic peptide (Figure 2A). This
mutant did not produce any 5-epi-aristolochene but, instead,
produced β-farnesene and farnesol as the major products
(Figure S4). TEAS mutants with more subtle active site
changes, including V277L, Y404F, L407I, and L512I, showed
alkylation on the V[442−453]K peptide, as well (Table 1 and
Figure S3). Unlike the W273E mutant, these mutants still
produced some 5-epi-aristolochene during the time course of
alkylation and inactivation (Figure S4). These results
demonstrate that active site mutations in a relatively product-
specific sesquiterpene synthase such as TEAS elicit self-
alkylation of a catalytically essential and highly conserved
2+
Mg -binding “DTE” motif of TEAS, while I[515−532]K
includes part of the J/K loop. This catalytic loop functions as a
19
dynamic active site lid upon substrate binding and ionization.
In general, self-alkylation occurred most readily for active site
mutants involving cation-stabilizing aromatic residues (Trp273
and Tyr404) (Figure 2A and Figures S2 and S3). The most
predominant self-alkylation occurred in the TEAS W273E
D
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TPS motif. Unexpectedly, alkylation did not occur on the
nucleophilic mutations originally designed to trap specific
carbocations.
truncation and the two additional stop codons, the gene
represents a loss-of-function TEAS homologue, but nonethe-
less, this sequence reveals a mutation at an active site position
we showed to cause self-alkylation upon even subtle structural
substitutions (Figure S3). The corresponding TEAS L407P
mutant, when expressed as a full length protein, showed
pronounced self-alkylation at Asp444 (Figure S7). This loss-of-
function TEAS gene homologue with a self-alkylation-
promoting mutation identified in the N. tabacum genome
suggests that self-alkylation can occur in the evolution of plant
sesquiterpene synthases and represents a significant constraint
during the natural evolution of TPSs.
Quantitative Self-Alkylation of TEAS W273E Yields a
2+
Farnesyl Ester of a Mg -Coordinating Aspartate. We
used protein X-ray crystallography to characterize both the site
of modification and the chemical structure of the alkylated
residue in TEAS. We focused on the W273E mutant of TEAS,
which yielded the highest level of farnesylation [∼50% for
tryptic peptide V[442−453]K (see Figure 2A)]. For compar-
ison, we obtained a crystal structure of nonalkylated TEAS
W273E refined to 2.3 Å resolution (PDB-entry: 5DHI, Table
S1). This “apo” TEAS crystal structure revealed several
pertinent chemical features, including the extended conforma-
Carbocation-Mediated Alkylation Results in Mecha-
nism-Based Enzyme Inhibition. To investigate the catalytic
consequences of inactivation of TEAS mutants by alkylation at
2
+
tion of the Glu273 side chain and a Mg ion coordinated to
the “DTE” motif, Asp444, Thr448, and Glu452 (Figure 2D). In
marked contrast, the crystal structure of alkylated TEAS
W273E refined to 2.43 Å resolution (PDB-entry: 5DHK, Table
S1) unmistakably revealed a bent conformation of Glu273, the
absence of Mg2 coordinated to the “DTE” motif, and, most
notably, an additional elongated stretch of electron density
contiguous with Asp444. This latter electron density is most
consistent with a linear farnesyl group covalently bound as an
ester (Figure 2C). The covalent linkage refined and displayed
unbiased electron density most reliably as a C1−farnesyl ester
bond linked to the carboxyl group of the Asp444 side chain in
2+
a Mg -binding site, the sesquiterpene products of TEAS
W273E and TEAS Y404F were characterized after incubation
with FPP. TEAS W273E was chosen because of its near
stoichiometric level of C15 alkylation (Figure 2A), and TEAS
Y404F was selected because it still produces the wild-type
product 5-epi-aristolochene (Figure S4). After incubation
(consistent with the physiological time course of TPS activity
in planta) with FPP for 12 h and protein purification, TEAS
W273E showed almost no sesquiterpene production compared
to the positive control (Table 2). Similarly, TEAS Y404F
+
2
7
phenix.refine.
Table 2. GC−MS Analyses of Product Yields of TEAS
Reactions with (E,E)-FPP Depending on the Alkylation
This site of alkylation, Asp444, within tryptic peptide
V[442−453]K, was further verified by electron transfer and
higher-energy collision dissociation tandem mass spectrometry
a
State
28
reaction time
(
EThcD-MS/MS) (Figure 2E and Figure S5). Alkylation
b
product yield (%) (main
within tryptic peptide I[515−532]K was not detected by
EThcD-MS/MS. However, the W280E mutant (equivalent to
TEAS W273E) of the orthologous TEAS-like TPS, premnas-
protein
0−12 h 12−24 h
product)
TEAS W273E
no FPP
FPP
FPP
100 (β-farnesene)
1 ± 0.3 (β-farnesene)
N/D
29
FPP
pirodiene synthase from Hyoscyamus muticus (HPS), yields an
alkylated tryptic peptide I[522−528]K consistent with the
alkylation of the tyrosine corresponding to TEAS-Tyr520
FPP
no FPP
FPP
TEAS Y404F
no FPP
FPP
100 (5EA)
FPP
86 ± 7 (5EA)
N/D
(Figure S6). Thus, on the basis of the high degree of sequence
FPP
no FPP
FPP
and active site homology of these two sesquiterpene
1
2,13
TEAS wild type
no FPP
FPP
100 (5EA)
synthases,
TEAS-Tyr520 and HPS-Tyr527 most likely
FPP
105 ± 11 (5EA)
N/D
represent additional alkylation sites in TEAS and HPS,
respectively, via a tyrosine farnesyl ether. In addition,
farnesylation of tyrosine via an electrophilic aromatic
substitution is possible. However, the tyrosine−farnesyl linkage
appears to be labile in the MS gas phase. There is a significant
mass loss attributed to the farnesyl moiety in the MS spectrum
FPP
no FPP
a
Analyzed TEAS constructs were TEAS W273E, TEAS Y404F, and
wild-type TEAS. Abbreviations: SD, standard deviation; 5EA, 5-epi-
b
aristolochene; N/D, not detected. Means ± the standard deviation,
reactions in triplicate (2.5 μM enzyme and 250 μM FPP).
(Figure 2B, spectrum 4). This experimental observation is most
consistent with the presence of a farnesyl−tyrosine ether
linkage rather than a farnesyl−tyrosine carbon−carbon bond.
Self-Alkylation of a Natural Mutation in a TEAS
Homologue Revealed by Genome Mining. Gene
duplication in plants often leads to the evolution of new
showed considerably reduced 5-epi-aristolochene production.
The reduction of product formation over a 12 h incubation
with FPP was ∼99% for TEAS W273E (β-farnesene as the
main product detected) and 7−21% for TEAS Y404F (5-epi-
aristolochene as the main product detected) (Table 2).
To test whether substrate-mediated alkylation of TEAS was
reversible, TEAS W273E and TEAS Y404F were incubated for
12 h with FPP, buffer exchanged to remove FPP and reaction
products, and incubated for an additional 12 h without FPP.
The corresponding hexane extracts of TEAS W273E and TEAS
Y404F incubated with FPP substrate showed no β-farnesene,
farnesol, or other putative hydrolyzed alkylation products,
indicating that the alkylation modification is stable and resistant
to recovery of catalytic activity. Additional tests with wild-type
TEAS demonstrated that the reduction in major product
production over time was not a result of enzyme degradation
3
0
chemotypes particularly in polyploid plants. To assess
whether self-alkylation-causing mutations can naturally occur
in sesquiterpene synthases, we searched the recently published
31
genome of tobacco strain Nicotiana tabacum Basma Xanthi
for TEAS homologues possessing active site changes (Figure
S7). Among six homologous TEAS genes identified, including
three genes with conserved active site residues and a putative
1
2,13
premnaspirodiene synthase (TEAS M9 mutant),
we
uncovered one highly homologous TEAS pseudogene with a
L407P mutation, a truncated N-terminus, and two internal stop
codons at positions 376 and 520. Because of the N-terminal
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Figure 3. Self-alkylation analysis of wild-type TEAS with (E,E)-farnesyl diphosphate analogues at different reaction temperatures. (A) Sesquiterpene
substrates tested in self-alkylation studies (Table 1). (B) Self-alkylation analysis of wild-type TEAS with sesquilavandulyl diphosphate (SPP). (C)
Self-alkylation analysis of wild-type TEAS with (E,E)-FPP at 25 and 42 °C. For detailed MS and MS/MS analysis, see Figure S8.
(
(
(
Table 2). The estimated alkylation levels of TEAS W273E
∼50%) and TEAS Y404F (∼5%) after FPP reaction for 12 h
Figure 2A and Figure S3) are not sufficiently high to account
temperatures can cause alkylation of wild-type TEAS during
substrate turnover. Alkylation levels were measured using the
conventional substrate (E,E)-FPP at 25, 37, and 42 °C. Wild-
type TEAS produced 5-epi-aristolochene as a main product at
all reaction temperatures (Figure S9). Alkylation was not
detected on Asp444 or on Tyr520 of TEAS at 25 or 37 °C.
However, at 42 °C, alkylation was readily detected on Asp444
(V[442−453]K) (Table 1, Figure 3C, and Figure S8),
demonstrating that self-alkylation can also be promoted by
increased reaction temperatures.
for the corresponding product yield reductions. This
discrepancy is likely due to both loss of alkylated peptides
during proteomic analysis and protein aggregation precipitated
by alkylated TEAS mutants. Although inactivation rates seem
low at first blush (Table S3), they result in a significant loss of
product yields as shown in Table 2 and, in the context of
multiple mutations, likely influence TPS active site evolution
during diversification and natural selection.
Increased Temperatures and Introduction of Sub-
strate Analogues Lead to Alkylation in Wild-Type TEAS.
Mechanism-based inhibition has been characterized in TPSs
with unnatural substrate analogues such as vinyl-farnesyl
Alkylation Analysis of Catalytically Specific and
Catalytically Promiscuous Sesquiterpene Synthases.
The alkylation resulting from mutational replacement of active
site residues or substitution of structurally diverse substrates
was investigated in other sesquiterpene synthases possessing
varying levels of product complexity. Three dominant-product
sesquiterpene synthases were employed: H. muticus premnas-
2
6
diphosphate. To show that natural substrate analogues can
also cause self-alkylation in TPSs, wild-type TEAS was
incubated for 12 h with sesquilavandulyl diphosphate (SPP),
an irregular sesquiterpene substrate employed during secondary
2
8
pirodiene synthase (HPS), citrus valencene synthase
3
5
(CVS), and Aspergillus terreus aristolochene synthase
32−34
36
metabolism of plants and marine microbes (Figure 3A).
(ATAS). In addition, three multiproduct sesquiterpene
Proteomic LC−MS analysis of wild-type TEAS after SPP
incubation detected alkylation on tryptic peptides V[442−453]
K and I[515−532]K (Table 1, Figure 3B, and Figure S8). The
GC−MS product profile of wild-type TEAS with SPP substrate
showed sesquilavandulene as the main product and sesquila-
vandulol and cyclized sesquilavandulenes as minor products
based on GC−MS spectra (Figure S9). The natural substrate
synthases were tested: Santalum spicatum santalene synthase
13
24
(SspiSSy), Abies grandis γ-humulene synthase (γHS), and
9
Pogostemon cablin patchoulol synthase (PAS). Alkylations by
active site changes were tested in all TPSs by mutations of
tryptophan or tyrosine residues structurally homologous to the
active site Trp273 in TEAS to glutamate or cysteine residues
(Figure 1A and Figures S10 and S11). Mutation of this site in
TEAS resulted in appreciable substrate-dependent alkylation.
Alkylations by natural (E,E)-FPP analogues were tested in
selected TPSs by incubation with SPP or (Z,E)-FPP (Table 1).
All dominant-product sesquiterpene synthases showed self-
alkylation with their conventional substrate FPP after active site
mutations (Table 1 and Figures S12 and S13). Incubation with
nonconventional substrates resulted in alkylation in wild-type
(
Z,E)-FPP (Figure 3A), with altered double bond stereo-
chemistry, resulted in alkylation of Tyr520 (Table 1 and Figure
S8). The characterized alkylation of wild-type TEAS with SPP
and (Z,E)-FPP shows that natural substrates of TPSs can also
induce alkylation in sesquiterpene synthases.
Because plants experience wide fluctuations in temperature
during day−night cycles, we tested whether increased reaction
F
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HPS but not in CVS (Table 1 and Figure S12). Multiproduct
sesquiterpene synthases showed little to no alkylation with FPP
upon active site mutations, and incubation with nonconven-
tional FPP substrates resulted in alkylation in only wild-type
PAS (Table 1 and Figures S13 and S14). Our alkylation
analyses of specific and promiscuous sesquiterpene synthases
suggest that dominant-product synthases are more prone to
self-alkylation upon active site mutations. In addition, the
detected alkylation of PAS after SPP incubation showed that
alkylation can also occur in multiproduct sesquiterpene
synthases with nonconventional substrates, although to a lesser
degree than observed in dominant-product synthases. Finally,
the alkylation of ATAS Y61C after (E,E)-FPP turnover, and the
alkylation of wild-type PAS with SPP, showed that
sesquiterpene synthases with an asparagine instead of an
cationic centers. The latter covalent modification of the TEAS
active site leads to enzyme inhibition.
These results suggest that enzymes employing chemically
reactive carbocation chemistry must balance the presence of
nucleophilic residues used for substrate turnover with the
unintended consequences of mechanism-based inactivation by
covalent modification of the active site. Given a recent study of
the evolutionary trajectory of sesquiterpene cyclization
39
promoted by epistatic active site mutations, it will be
interesting to assess the degree to which cyclization-inducing
mutations favored by natural selection also limit self-alkylation
in comparison to mutations suggested to favor more ancient
noncyclizing activity.
Previous studies hypothesized that cationic intermediates of
the catalytic cascade reactions of TPSs might be trapped in
2
+
25
aspartate in the “NSE/DTE” motif for Mg binding can also
undergo alkylation, implying alkyl asparagine amides as putative
alkylation products.
active sites by nucleophilic residues. Our investigation of self-
alkylation in TEAS is, to the best of our knowledge, the first
study to confirm this hypothesis and to identify two conserved
catalytic motifs acutely sensitive to farnesyl cation-based
covalent modifications. Moreover, multiple alkylated species
were detected at two active site motifs. The J/K loop lid
peptide, in particular, showed multiple species based on
different retention times of alkylated peptides during LC-
based separation (Figure 2A and Figure S2). In several TEAS
point mutants undergoing alkylation, including V277L, Y404F,
L407I, and L512I, 5-epi-aristolochene is formed, suggesting that
structurally distinct carbocations may also undergo quenching
by other nucleophiles positioned proximal to cationic centers,
including Thr448 and Asp525. Ongoing studies will address the
structural identity of captured isoprenoid intermediates by
hydrolytic release of these trapped moieties from alkylated
TPSs.
DISCUSSION
■
In this study, the occurrences, locations, chemistries, and
inhibitory consequences of mechanism-based alkylations of
specific and promiscuous sesquiterpene synthases were
investigated. Alkylation assays relied on bottom-up proteomic
LC−MS analyses of sesquiterpene synthases by monitoring the
mass shifts of covalently modified enzyme side chains on tryptic
+
peptides by C15 carbocations (C H ; Δm = 204.188 Da).
1
5
24
Initial complications associated with the detection of alkylated
peptides with protected proteolytic cleavage sites, e.g., CVS,
γHS, and PAS, and with cleavage sites residing in the “NSE/
DTE” motif or on the active site lid as seen in γHS (Figure
S13) were addressed using contrasting solid phases for LC−MS
separation and analyses. Accurate quantification of substrate-
mediated TPS alkylation in this study by LC−MS was not
possible because of differences in tryptic cleavage efficiencies
between nonalkylated and alkylated TPSs, loss of alkylated
peptides during sample preparation and separation, and
variation in ionization efficiencies between nonalkylated and
alkylated peptides. However, our LC−MS method is a relatively
facile starting point for estimating the extent of substrate-
mediated alkylation levels in more complex samples such as
those found in elicited plant proteomes during the course of
terpene biosynthesis. Ongoing studies are aimed at quantitation
Substrate-mediated alkylations were readily detected in active
site mutants of dominant-product TPSs but were rarely
detected in multiproduct TPSs. Multiproduct TPSs may direct
mechanisms possessing more conformational freedom for
initially formed farnesyl cations. In turn, these dynamic TPS
scaffolds provide more alternative pathways during the cationic
rearrangements leading to additional sesquiterpene prod-
9
,24
ucts.
This level of catalytic promiscuity in cationic reaction
pathways mediated by the active site of multiproduct TPSs may
have evolved, in some instances, not only to produce a mixture
of advantageous products, but to also avoid covalent cation
capture. Avoidances of premature enzyme inhibitions during
the transformations of isoprenoid diphosphates in TPSs likely
serves as additional hitherto unappreciated constraints during
the evolution of TPS-based natural product biosyntheses. The
large structural diversity of cyclic scaffolds found in
sesquiterpene natural products is principally determined by
the stereo- and regiochemistry of cationic transformations
1
4
37
of alkylation using C-labeled substrates and quantitative
3
8
proteomics. Nevertheless, ETD-MS/MS yielded a low-
abundance z-ion series that confirmed the Asp444 alkylation
site (Figure S5).
The mechanism-based alkylations of sesquiterpene synthases
2+
at Mg -binding “NSE/DTE” motifs and along active site
catalytic loops result from early linear farnesyl cation captures
instead of alkylation by cyclic carbocations formed during latter
parts of the cation-based cascade mechanisms. For example,
TEAS W273E still binds and ionizes FPP, but the active site
mutation disfavors formation of the germacryl cation (Figure
S1) because of the loss of the tryptophan−π-cation interaction.
The result is reduced production of cyclic products in TEAS
W273E compared to that in wild-type TEAS. Instead of
intramolecular cyclization, the farnesyl cation can first undergo
rapid proton loss to yield the main product β-farnesene.
Second, the farnesyl cation can be quenched by water to yield
farnesol as a minor product. Third, the farnesyl cation can be
captured by attack of active site nucleophiles, including the side
chains of Asp444 and Tyr520 buttressing the farnesyl−C1
40
catalyzed by class I TPSs. To facilitate specific cationic
transformations leading to bioactive terpene structures, TPSs
must evolve to promote cationic reaction cascades while
avoiding the premature enzyme-based capture of these cations
and ensuing inactivation. These hypotheses are supported by
the observations that none of the investigated wild-type TPSs
showed significant levels of self-alkylation with the conventional
substrate (E,E)-FPP at room temperature (Table 1, Figure 3C,
and Figures S11 and S13). During the evolution of terpene
biosynthesis, enzyme inhibition by self-alkylation may be
limited by maintaining large active site volumes, such as
those found in multiproduct sesquiterpene synthases. In
contrast, the evolution of increased product specificity often
G
DOI: 10.1021/acschembio.5b00539
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
results in smaller and more specialized active sites that appear
to be more susceptible to substrate-based alkylation upon
subtle mutational changes. In short, alkylations may serve as
constraints during the evolution of other enzymes employing
above, and then incubated with (E,E)-FPP (250 μM) in reaction buffer
for 12 h at 25 °C (reaction time of 12−24 h, 2.5 μM enzyme). The
reaction mixtures were then extracted with hexane and analyzed by
4
6
GC−MS as described previously. For analysis of putative hydrolysis
of enzyme alkylation modifications, wild-type TEAS, TEAS W273E, or
TEAS Y404F was incubated with (E,E)-FPP for 12 h at 25 °C
2
+
cationic mechanisms, Mg ions and carboxyl-bearing side
1
1
chains for isoprenoid diphosphate ionization. Finally,
mechanism-based self-alkylation may underlie metabolic
diseases associated with mutations in pervasive terpene
(
reaction time of 0−12 h), buffer exchanged as described above, and
then incubated without substrate in reaction buffer for 12 h at 25 °C
reaction time of 12−24 h, 2.5 μM). The reaction mixtures were then
extracted with hexane and analyzed by GC−MS as described
(
41
biosynthetic genes found across all three domains of life.
4
6
The propensity for cation-based alkylation of TPSs is also an
important consideration for the metabolic engineering of high-
value terpene production in heterologous hosts. Metabolic
engineering of host organisms for biotechnological production
previously. Assays for inhibition analyses were conducted in triplicate
with 4-chlorotoluene (Sigma-Aldrich) as an internal standard.
Sesquiterpene products were quantified by autointegration of product
peaks in GC−MS total ion chromatograms with Agilent Chemstation
software (G1701DA). Kinetic analyses of TEAS W273E and TEAS
42
of sesquiterpenes has advanced considerably. However, the
optimization of TPSs or engineering of new TPS activities has
failed to account for the possibility of mechanism-based TPS
4
6
Y404F were conducted as described previously.
Alkylation Analysis of Sesquiterpene Synthases by Bottom-
Up Proteomics. Sesquiterpene synthases (2.5 μM) were incubated
with substrate [250 μM, (E,E)-FPP, (Z,E)-FPP, or SPP] in reaction
43,44
inactivation through active site alkylation.
Understanding
the correlations between the extent and regiochemistry of
alkylations of TPSs with mutations and substrate alterations
should facilitate TPS optimizations through rapid alkylation
analyses of mutant libraries. Future studies will focus on in vivo
characterization of self-alkylation of sesquiterpene synthases in
plant cells and expression hosts used commercially, and
additional enzymes exposed to reactive carbocations that
biosynthesize terpene natural products.
buffer [50 mM Bis-Tris propane and 15 mM MgCl (pH 7.5)] for 12 h
2
at 25 °C. For investigation of reaction temperature effects, wild-type
TEAS was also assayed at 37 and 42 °C. The reaction mixtures were
adjusted to pH 8 with 1 M Tris-HCl (pH 8), with 1 μg of trypsin
(
Trypsin singlets, Sigma-Aldrich) added, and incubated for 2−6 h at
5 °C. The tryptic digests were analyzed by liquid chromatography−
tandem mass spectrometry (LC−MS/MS) on a PROTO300 column
C4, 5 μm, 250 mm × 4.6 mm, Higgins Analytical, Inc.) connected to
2
(
an Agilent 6530 Accurate-Mass Q-TOF LC/MS instrument in positive
ion mode. The LC gradient of solvent A (acetonitrile and 0.1% formic
acid) and solvent B (0.1% formic acid) was as follows: 10% A (0 min),
METHODS
■
1
1
0% A (10 min), 30% A (15 min), 70% A (55 min), 100% A (60 min),
Chemicals and Primers. Chemicals were obtained from Sigma-
Aldrich, if not noted otherwise. LC−MS solvents were Honeywell
LabReady blends for LC−MS application. All DNA primers were
obtained from IDT DNA Technologies. (E,E)-FPP and (Z,E)-FPP
were obtained from Isoprenoids, LLC. SPP was obtained by synthesis
as described previously and provided by the Moore lab (University
of California at San Diego, La Jolla, CA).
00% A (63 min), 10% A (64 min), and 10% A (70 min) at a flow rate
−1
of 0.7 mL min . MS parameters were as follows: time segment 1, 0−
0 min (LC flow to waste); time segment 2, 10−70 min (LC flow to
MS). Source parameters were as follows: gas temperature, 350 °C; gas
1
−1
45
flow, 8 L min ; nebulizer, 35 psig; positive ion polarity. Scan source
parameters were as follows: VCap, 3500 V; Fragmentor, 175 V;
Skimmer1, 65 V; OctopoleRFPeak, 750 V; Acquisition mode
AutoMS2, MS range of m/z 400−1700, MS scan range of 2
spectra/s, MS/MS scan range of 3 spectra/s, MS/MS isolation
width of m/z 4, collision energy 30 eV, Precursor Selection: Static
Exclusion Range 100−500 m/z, MS Abs. threshold 200, MS Rel.
threshold 0.01%, MS/MS Abs. threshold 500, MS/MS Rel. threshold
Sesquiterpene Synthase Constructs. TPS-encoding genes
TEAS, TOBSQPC; HPS, XXU20187; CVS, GQ988384; SspiSSy,
(
HQ343278; γHS, U92267; PAS, AY508730) were expressed and
mutated from pET28a constructs harboring the corresponding genes
with an N-terminal His tag (nine-His tag, PAS; eight-His tag, TEAS
and HPS; six-His tag, SspiSSy, γHS, and CVS) and N-terminal
thrombin cleavage site before the start codon of the terpene synthase
gene. Terpene synthase mutants were constructed by site-directed
mutagenesis based on the QuikChange site-directed mutagenesis kit
0
.01%. Q-TOF MS data were analyzed with Agilent MassHunter
software (B.05.00).
Alkylation Analysis of Sesquiterpene Synthases by EThcD-
MS/MS. Samples were precipitated with methanol and chloroform.
Dried pellets were dissolved in an 8 M urea/100 mM Tris-HCl (pH
(
Agilent) using designed mutagenesis primers (Table S2).
Protein Expression and Purification. See the text of the
8
.5) mixture. Proteins were reduced with 5 mM tris(2-carboxyethyl)-
Supporting Information.
phosphine hydrochloride (TCEP) (Sigma-Aldrich) and alkylated with
10 mM iodoacetamide (Sigma-Aldrich). Proteins were digested
overnight at 37 °C in a 2 M urea/100 mM Tris-HCl (pH 8.5)
mixture with trypsin (Promega). Digests were quenched with formic
acid, at a final concentration of 5%. The digested samples were
analyzed on a Fusion Orbitrap tribrid mass spectrometer (Thermo).
The digests were injected directly onto a 20 cm, 100 μm inside
diameter column packed with 5 μm ODS-AQ C4, C8, or C18 resin
(YMC). Samples were separated at a flow rate of 400 nL min− on an
Easy nLC2 instrument (Thermo). Buffers A and B were 0.1% formic
acid in water and acetonitrile, respectively. A gradient of 1 to 10% B
over 10 min, an increase to 45% B over 70 min, an increase to 100% B
over an additional 50 min, and a hold at 90% B for a final 10 min of
washing was used before returning to 1% B. The column was re-
equilibrated with 10 μL of buffer A prior to the injection of samples.
Peptides were eluted directly from the tip of the column and
nanosprayed directly into the mass spectrometer by application of a
voltage of 2.5 kV at the back of the column. The Orbitrap Fusion was
operated in a data-dependent mode. Full MS1 scans were collected in
the Orbitrap at 120K resolution with a mass range of m/z 400−1600
Sesquiterpene Synthase Assays and Product Profiling by
GC−MS. Sesquiterpene synthases (2.5 μM) were incubated with
substrates [250 μM, (E,E)-FPP, (Z,E)-FPP, or SPP] in reaction buffer
[
50 mM Bis-Tris propane and 15 mM MgCl (pH 7.5)] for 12 h at 25
C. The reaction mixtures were subsequently extracted with hexane by
2
°
being vortexed for 30 s and the hexane layers were analyzed by GC−
45
MS as described previously. For investigation of reaction temper-
ature effects, wild-type TEAS was also assayed at 37 and 42 °C.
For inhibition analysis, wild-type TEAS, TEAS W273E, or TEAS
Y404F (2.5 μM) was incubated with (E,E)-FPP (250 μM) in reaction
buffer for 12 h at 25 °C (reaction time of 0−12 h). The reaction
mixtures were then concentrated using 30 kDa cutoff protein
concentrators (Millipore) and washed three times with reaction
buffer. Subsequently, buffer-exchanged wild-type TEAS, TEAS
W273E, or TEAS Y404F (2.5 μM) was incubated again with (E,E)-
FPP (250 μM) in reaction buffer for 12 h at 25 °C (reaction time of
1
1
2−24 h). The reaction mixtures were then extracted with hexane and
46
analyzed by GC−MS as described previously. For comparison of
enzyme inhibition by the first reaction period, wild-type TEAS, TEAS
W273E, or TEAS Y404F (2.5 μM) was incubated without substrate for
5
1
2 h at 25 °C (reaction time of 0−12 h), buffer exchanged as described
and an AGC target of 5e . The cycle times were set to 5 s, and within
H
DOI: 10.1021/acschembio.5b00539
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
this 5 s interval, the most abundant ions per scan were selected for
homologues, and sequence alignment of analyzed TPSs
either CID or EThcD and detected in the orbitrap with a resolution of
(PDF)
5
1
5K with an AGC target of 5e and a minimal intensity of 5000.
Structure factor and coordinate files of the reported
structures are available from the Protein Data Bank
Maximal fill times were set to 250 and 100 ms for MS and MS/MS
scans, respectively. Quadrupole isolation at m/z 3 was used;
monoisotopic precursor selection was disabled, and dynamic exclusion
was used with an exclusion duration of 30 s. Orbitrap MS data were
analyzed with QualBrowser software from the Thermo Xcalibur
package (version 2.1).
(
5DHI and 5DHK).
AUTHOR INFORMATION
Corresponding Author
E-mail: noel@salk.edu.
■
Genome Mining of N. tabacum for Tobacco 5-epi-
*
Aristolochene Synthase Homologues. The genome of N. tabacum
31
str. Basma Xanthi (GenBank entry AWOK01000000) was searched
Notes
for gene homologues of tobacco 5-epi-aristolochene synthase
The authors declare no competing financial interest.
19
(
(
GenBank entry TOBSQPC) with blastn. Highest blastn hits
>80% identity) were translated and analyzed for completeness of
ACKNOWLEDGMENTS
■
the catalytic C-terminal domain sequence and for active site residues
12,13
We thank G. Louie for training in X-ray crystallography and H.-
J. Koo for GC−MS training and providing a TEAS model for
initial molecular refinement. We thank B. Moore (University of
California at San Diego) for mass spectrometry instrument
access, S. Diethelm (University of California at San Diego) for
providing synthetic SPP, and J. Bohlmann and J. Chappell for
discussions and TPS genes. J.P.N. is an Investigator of the
Howard Hughes Medical Institute. R.D.K. is a Howard Hughes
Medical Institute Fellow of the Life Sciences Research
Foundation. This research was funded by the National Science
Foundation (EEC-0813570 to J.P.N.) and the National
Institute of General Medical Sciences (8 P41 GM103533 to
J.R.Y.III). The Berkeley Center for Structural Biology is
supported in part by the National Institutes of Health, National
Institute of General Medical Sciences, and the Howard Hughes
Medical Institute. The Advanced Light Source is supported by
the Director, Office of Science, Office of Basic Energy Sciences,
of the U.S. Department of Energy under Contract DE-AC02-
and M9 residues.
Protein Crystallization. For preparation of farnesylated protein
for crystallization trials, TEAS W273E (2.5 μM) was incubated with
FPP (250 μM) for 18 h at 25 °C in reaction buffer [50 mM Bis-Tris
propane and 15 mM MgCl (pH 7.5)], and the treated protein was
2
then transferred to a 30 kDa protein concentrator (Millipore), washed
three times with protein buffer [50 mM Tris-HCl, 100 mM NaCl, and
2
mM DTT (pH 8)], and finally brought to a concentration suitable
for crystallization. Alkylated and untreated TEAS W273E samples
were crystallized by the hanging-drop vapor-diffusion method with
−1
drops containing a 1:1 ratio of a protein solution (10−12 mg mL )
and a crystallization solution [0.1 M MOPSO (pH 7), 100 mM
MgOAc, and 14% (w/v) PEG 8000]. Tetragonal crystals were
observed within 1 day. For X-ray data collection, crystals were flash-
frozen in liquid nitrogen in a crystallization solution containing 20%
(
v/v) glycerol.
X-ray Crystallographic Analyses of TEAS Active Site Mutant
W273E. X-ray diffraction data were obtained from frozen crystals at
beamline 8.2.1 of the Advanced Light Source, Lawrence Berkeley
National Laboratory (Berkeley, CA). Diffraction images were collected
on an ADSC Quantum 315R CCD detector and indexed and
0
5CH11231.
47
integrated with iMOSFLM, and measured diffraction intensities were
48
49
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Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.5b00539.
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