Characterization of arylalkylamine n-acyltransferase from tribolium castaneum: an investigation into a potential next-generation insecticide target

Article abstract of DOI:10.1021/acschembio.9b00973

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Full text of DOI:10.1021/acschembio.9b00973

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Article
Characterization of Arylalkylamine N-Acyltransferase from Tribolium
castaneum: An investigation into a potential next-generation insecticide target
Brian G. O'Flynn, Eric M. Lewandowski, Karin Claire Prins, Gabriela Suarez,
Angelica N. McCaskey, Nasha M. Rios-Guzman, Ryan L. Anderson, Britney
A. Shepherd, Ioannis Gelis, James W. Leahy, Yu Chen, and David J. Merkler
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00973 • Publication Date (Web): 22 Jan 2020
Downloaded from pubs.acs.org on January 27, 2020
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Characterization of Arylalkylamine N-Acyltransferase from
Tribolium castaneum: An investigation into a potential next-
generation insecticide target
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†
‡
†
†
Brian G. O’Flynn , Eric M. Lewandowski , Karin Claire Prins , Gabriela Suarez , Angelica N.
†
†
† ∥
†
†
McCaskey , Nasha M. Rios-Guzman , Ryan L. Anderson , Britney A. Shepherd , Ioannis Gelis ,
§
† ‡
‡
†
James W. Leahy , Yu Chen , & David J. Merkler*
†
‡
Department of Chemistry, University of South Florida, Tampa, Florida, 33620, United States
Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, 33612, United
States
§
Center for Drug Discovery and Innovation, University of South Florida, Tampa, Florida, 33620, United States
Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus,
Aurora, CO 80238.
∥
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Abstract
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The growing issue of insecticide resistance has meant the identification of novel insecticide targets
has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been
suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation
of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism,
hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides.
The unique nature of each AANAT isoform characterized indicates each organism accommodates
an assembly of discrete AANATs relatively exclusive to that organism. This implies a high
potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented
here is a thorough kinetic and structural analysis of AANAT found in one of the most common
secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named
TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-
CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning as the role of acyl-donor.
Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the
kinetic and chemical mechanism of catalysis. The kinetic mechanism is ordered sequential
mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies
identified amino acids critical to catalysis, providing insights about the chemical mechanism of
TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing
valuable information about its active site. This combination of kinetic analysis, and
crystallography, alongside mutagenesis, and sequence analysis shines light on some approaches
possible to target TcAANAT0 and other AANATs for novel insecticide design.
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Introduction
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The agricultural industry relies extensively on pesticides in an effort to prevent crop and stored
grain destruction. The increased demand for crop protection through pesticide application is
expected to drive the global agrochemicals market to $308.92 billion by 2025.1 The issue of
pesticide resistance, however, has meant that as a species develops ways to negate the effects of a
pesticide, an increased dose must be applied.2 While it is argued that high doses are necessary for
complete control over a crop,3,4 this has led to these chemicals entering our food supply at
dangerous levels. Many of these are known or probable carcinogens,3,8 harming not just
mammals,7,8 but also important pollenating species such as the honey bee.9–11 With regards to
insect control, many novel insecticides have entered the market with lower toxicity and
selectivity;12,13 however, their low polypharmacology classically means they are not as
economically viable as commercial insecticides, potentially leading to an increased rate of
resistance development.14
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To combat insecticide resistance, a combination of (1) mixing, rotating, and mandatory
breaks in use of currently marketed insecticides, (2) continued design of novel insecticides, and
(3) identification of novel enzymatic targets for insecticides must be utilized. A thorough
understanding of the mechanism of action of a potential new target will enable the development
of inhibitors that will target just one, or a select group of enzymes within a specific insect pest.
This will reduce the negative secondary effects on mammals and/or pollenators.
Arylalkylamine N-acyltransferase (often denoted arylalkylamine N-acetyltransferase –
AANAT) has shown potential as a novel insecticide target.15,16 These enzymes catalyze the transfer
of an acyl group from an acyl-CoA donor to a primary amine acceptor: R1-NH2 + R2-CO-S-CoA
→ R2-CO-NH-R1 + CoA-SH. This reaction is synonymous with gene transcription regulation via
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histone acetylation (histone acetyltransferase – HAT),17,18 mammalian circadian rhythm
maintenance via serotonin acetylation (serotonin N-acetyltransferase – SNAT), 19,20 and dopamine
acetylation, a key step in insect cuticle sclerotization and melanism21,22 (dopamine N-
acetyltransferase – DAT).23–25 Additionally, insects lack monoamine oxidase, a regulatory enzyme
which facilitates the inactivation and excretion of monoamine neurotransmitters, such as dopamine
and norepinephrine.26 AANAT is one of a group of enzymes proposed to perform this role in
insects.27,28 Recent evidence also suggests AANATs have a role in the biosynthesis of long-chain
fatty acid amides in insects.29,30
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As opposed to mammal and avian genomes, where only one AANAT has been identified
(SNAT),19,31 insects contain several iAANATs and iAANAT-like (iAANATL) enzymes, likely
due to the wide range of functions ascribed to the iAANATs in vivo. For example, Drosophila
melanogaster and Aedes aegypti have at least eight and thirteen iAANATs/iAANATLs,
respectively.32,33 Notably, sequence identity between the iAANATs/iAANATLs is most often
<40%.34 The low sequence homology combined with the variety of cellular functions of the
iAANATs/iAANATLs suggests these enzymes are excellent targets for novel insectide design.17,18
Thorough insight on the mechanistic and structural characteristics of an iAANAT will
foster the development of a specific insecticide targeted against that enzyme. This information will
allow us to predict structural features that can be built into a putative inhibitor, and to predict how
an enzyme may naturally evolve resistance to the inhibitor. Herein, we present an analysis of one
iAANAT from Tribolium castaneum, one of the most common secondary pests that infest and
destroy stored grain products worldwide. To date, insecticides such as phosphine, cyanogens,
chloropyrifos, and malathion have shown limited success at negating the damage caused by T.
castaneum.35–38 These pesticides are not viable long-term solutions for T. castaneum control
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because of emerging resistance (especially in developing countries) and the detrimental effects
these have on the environment.39
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Previous work on iAANATs has significantly expanded our understanding of the structure
and mechanisms (kinetic and chemical) of these enzymes; however, we are only now beginning to
appreciate their variability. Our data show TcAANAT0 (NCBI accession number: XM_967780.4)
continues this trend of heterogeniety with respect to kinetic and chemical mechanism, amine
acceptor specificity, acyl-CoA donor chain length, and structure/function characteristics. These
appreciable differences highlight the individuality of each AANAT, not just from insect to insect,
but also from isoform to isoform within each insect.
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Results and Discussion
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Acyl-CoA steady-state kinetic constants
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A panel of twelve acyl-CoAs were selected to test for activity with TcAANAT0: acetyl-,
propionyl-, butyryl-, DL--hydroxybutyryl, crotonyl-, and decanoyl-CoA all represented short-
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chain acyl-CoAs. DL--hydroxybutyryl CoA and crotonyl CoA were used to examine effect of -
hydroxylation and desaturation on activity, respectively. Palmitoyl, stearoyl- and oleoyl-CoA all
represented long-chain acyl-CoAs. Finally, malonyl-CoA and succinyl CoA represented
carboxylate-containing CoAs, and benzoyl-CoA represented an aryl CoA. Short-chain acyl-CoAs
were substrates for TcAANAT0 (Table 1), while the long-chain acyl-CoAs and malonyl-CoA were
not. Weak activity was also seen for succinyl-CoA, and benzoyl-CoA. Butyryl-CoA was shown to
have an ̴160-fold decrease in activity when -hydroxylated but only a 3-fold decrease when
unsaturated. Hydroxylation had the greatest effect on acyl-CoA binding, with the main reason for
the loss in activity being a sharp increase in KM. Interestingly, a trend of steadily decreasing KM
values from acetyl- to decanoyl-CoA was observed, which hints at an active site capable of
accepting and binding to longer chain CoAs. We have observed a similar trend for iAANATs from
Drosophila melanogaster and Bombyx mori.40,41 However, the decreasing kcat value as the length
of the acyl chain increases implies that catalysis is not feasible for long-chain acyl-CoA substrates.
This likely results from the amine binding pocket becoming sterically occupied upon long-chain
acyl-CoA binding, preventing either (a) the amine from binding, (b) the occurrence of a
catalytically important conformational change, or (c) improper positioning of the substrates within
the active site. Further investigation is needed to uncover the evolutionary and structural
divergencies that differentiate iAANATs that accept short-chain acyl-CoAs as substrates from
those that accept long-chain acyl-CoA as substrates, such as DmAANATL2 and BmAANAT.29,30
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Table 1: Steady-state kinetic constants of active acyl-CoA substrates for TcAANAT0. Kinetic constants were
measured using 300 mM Tris-HCl, pH 8.0, 375 M DTDP, and 5 mM tryptamine.
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kcat/KM
Substrate
Structure
kcat (s-1)
KM (M)
(M-1s-1)
O
(1.2 ± 0.1) ×
106
Acetyl-CoA
25 ± 1.8
29 ± 0.6
CoA
CoA
CoA
CoA
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H3C
C2H5
C3H7
C3H5
S
O
O
O
(8.0 ± 0.8) ×
105
Propionyl-CoA
Butyryl-CoA
21 ± 2.1
15 ± 1.8
17 ± 0.5
5.5 ± 0.1
S
(3.7 ± 0.5) ×
105
S
(1.1 ± 0.2) ×
105
Crotonyl-CoA
29 ± 3.5
3.3 ± 0.2
S
4:1(2E)
O
(1.0 ± 0.4) ×
103
(2.3 ± 0.1) ×
103
DL--hydroxybutyryl-
CoA
2.3 ± 0.5
0.8 ± 0.1
1.1 ± 0.1
CoA
HOC3H6
S
O
(9.6 ± 2.2) ×
105
Decanoyl-CoA
Succinyl-CoA
0.9 ± 0.2
CoA
C9H19
S
O
(6.8 ± 1.4) ×
102
(1.6 ± 0.3) ×
103
CoA
HOOCC2H4
S
O
CoA
(9.3 ± 1.3) ×
102
Benzoyl-CoA
48 ± 6.5
0.04 ± 0.01
S
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Amino steady-state kinetic constants
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A panel of >40 amines were screened against acetyl- and oleoyl-CoA to establish the amine
substrate specificity and to determine if the length of the acyl-chain of the acyl-CoA substrates
influences the specificity for the amine. A modified version of the strategy described by Dempsey
et al.41 was employed to evaluate the amines as TcAANAT0 substrates. The amines used in the
screening strategy included amino acids, an array of biogenic catecholamines and indolamines,
polyamines, and aminoglycosides. We also included amines with a pKa <8.0, i.e. amines that will
possess a deprotonated amine group at the pH used for our screening assay (pH 8.0). Those amines
demonstrating activity above the baseline activity level resulting from the non-enzymatic
hydrolysis of the acyl-CoA substrate were analyzed to determine their kinetic constants. No
activity was observed for any of the amines with oleoyl-CoA as the acyl donor; however, activity
was found for 10 amines paired with acetyl-CoA, summarized in Table 2. It should be noted that
the assay we employed to measure activity was dependent upon the release of free CoA-SH.42
Thus, the data of Tables 1 and 2 do not exclude the possibility of an amine-activited thioesterase
activity for TcAANAT0. Mass spectrometry was used to characterize the product (N-
acetyltryptamine) and confirm TcAANAT0 was, indeed, synthesizing acylated amines in vitro
(Table S1). The promiscuity of TcAANAT0 compared to the putative T. castaneum DAT
previously studied by Noh et al.43 indicated a more functionalized enzyme, providing a unique
insight into AANAT activity.
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Table 2: Steady-state kinetic constants of most active amine substrates for TcAANAT0. Kinetic constants were
measured using 300 mM Tris-HCl, pH 8.0, 375 µM DTDP, and 250 M acetyl-CoA
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kcat/KM
Substrate
Struture
kcat (s-1)
KM (M)
(M-1s-1)
NH2
(7.1 ± 1.1)
× 105
2-Phenethylamine
140 ± 18
100 ± 8.1
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HO
HO
(4.4 ± 0.3)
× 105
Dopamine
140 ± 11
340 ± 46
61 ± 1.3
78 ± 4.5
NH2
F
2,2’-Difluoro-2-
phenethylamine
(DFPE)
(2.3 ± 0.3)
x 105
NH2
F
HO
HO
(1.1 ± 0.1)
x 105
DL-Norepinephrine
480 ± 46
480 ± 77
53 ± 1.8
35 ± 2.0
NH2
OH
H
N
(7.4 ± 0.1)
x 104
Serotonin
Histamine
Tryptamine
HO
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NH2
N
(3.4 ± 0.4)
x 104
1,700 ± 170 57 ± 1.7
NH2
H
N
(9.2 ± 0.8)
x 104
260 ± 29
520 ± 61
33 ± 0.8
36 ± 1.3
NH2
HO
(7.0 ± 0.8)
x 104
Tyramine
Agmatine
NH2
H
H2N
N
(6.4 ± 0.6)
x 104
NH2
1,200 ± 110 77 ± 3.0
2,500 ± 140 43 ± 0.8
NH
OH
NH2
(1.7 ± 0.1)
x 104
Octopamine
HO
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TcAANAT0 kinetic mechanism
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The kinetic mechanism for TcAANAT0 was examined using a combination of initial velocity
kinetics analysis, dead-end inhibition, and direct ligand binding by isothermal calorimetry. A
kinetic fingerprint was obtained by varying the concentration of one substrate and measuring the
rates at several fixed concentrations of the other (Figure 1). The data was best fit to an ordered
bi-bi mechanism (2 = 27.5) as opposed to any variant of a random or non-sequential bi-bi
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mechanism (2 ≥ 36.2), which has been attributed to several AANATs, previously.40,44
Catalysis, in the case of TcAANAT0, involves the initial binding of acetyl-CoA and only takes
place after the formation of a TcAANAT0•acetyl-CoA•tryptamine ternary complex. ITC
confirmed this, with acetyl-CoA binding to free TcAANAT0 with a Kd vaule of 0.33 ± 0.04 M
(Figure S1). This is in good agreement with the Kd obtained for the binding of acetyl-CoA to Bm-
iAANAT3, 0.66 ± 0.03 .45
Next, to determine the order of substrate binding and product release, a set of dead-end
inhibition experiments was performed. The substrate analogs desulfo-CoA and tryptophol were
used as dead-end inhibitors. Inhibition constants were measured for all compounds. These data are
summarized in Figure 2 where Ki,s is the inhibition constant affecting the slope (when I binds to
E), and Ki,i is the inhibition constant affecting the intercept (when I binds to ES). The inhibition
patterns for desulfo-CoA, competitve vs. acetyl-CoA and non-competitive vs. tryptamine, and for
tryptophol, competitive vs. tryptamine and uncompetitive vs. acetyl-CoA, confirm an ordered
sequential mechanism for substrate binding, with acetyl-CoA binding first. Product inhibition was
attempted with N-acetyltryptamine, but no inhibition was detected at a concentration as high as 10
mM. N-Acetylagmatine was also readily available for testing, therefore, since TcAANAT0 utilizes
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agmatine as an amine substrate, it was also evaluated as a product inhibitor. However, with a
measured IC50 >50 mM, it was concluded that the acetylated amine products simply do not bind
to the free form of the enzyme, suggesting a conformational change upon acetyl-CoA binding.
This conformational change has been shown previously in Bombyx mori iAANAT to facilitate
interaction with the incoming amine.45 The inability to measure any significant inhibition with
either N-acetyltryptamine and N-acetylagmatine complicated attempts to conclusively define the
order of product release. However, ordered product release with the N-acetylamine being released
first and free CoA-SH being released second has been observed for other iAANATs and is
consistent with both the lack of any appreciable binding of the N-acetylamines to free enzyme, and
the ability of desulfo-CoA to do so.41
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Finally, we interrogated the TcAANAT0-catalyzed reaction using the bisubstrate analog,
CoA-S-acetyltryptamine, as an inhibitor. The observance of multiple rates of product formation,
rather than one initial steady-state rate followed by a plateau indicated slow-binding inhibition.
The true KI value (Kitrue) was found to be 16 ± 1.7  following the methods of Schlesinger et
al.46 and Merkler et al.47. This data is summarized in Table S2 and the mechanism of inhibition is
shown in Figure S4. This time-dependent inhibition via bisubstrate analog indicates a structural
reorganization upon binding of both substrates. This is akin to a kinetic proofreading mechanism,
wherein the active site affinity for the substrates increases upon intermediate formation, slowing
the reaction down and assuring correct product formation.48
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Figure 1: Acetyl-CoA and tryptamine initial velocity double reciprocal plots. (A) Velocities measured at fixed
concentrations of tryptamine: 720 M (), 360 M (), 125 M (), 75 M () and 50 M (). (B)
Velocities measured at fixed concentrations of acetyl-CoA: 100 M (), 37 M (), 20 M (), and 10 M
(). These data are best fit to the rate equation for a sequential mechanism (2 is 27.5 for Equation 2)
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Varied
Substrate
Inhibitor
Pattern
Inhibitor
Ki,s (mM)
Ki,i (mM)
Desulfo-CoA
Tryptophol
Desulfo-CoA
Tryptophol
NC
C
1.1 ± 0.2
0.3 ± 0.03
2.2 ± 0.1
--
0.6 ± 0.07
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9
Tryptamine
Acetyl-CoA
--
C
--
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UC
4.0 ± 0.2
H
N
CoA CH3
OH
Figure 2: Inhibition patterns observed for inhibitors of the acyl-CoA binding pocket (desulfo-CoA) and amine
binding pocket (tryptophol). Structures are shown for desulfo-CoA (left) and tryptophol (right). Kinetic constants
were measured with varied tryptamine concentrations of 50, 75, 100, 150, 300, and 500 M and fixed 50 M acetyl-
CoA. Acetyl-CoA concentrations were varied at 10, 20, 30, 50, 80, and 100 M with fixed 300 M tryptamine.
Tryptophol concentrations were varied at 0, 2 and 4 mM. Desulfo-CoA concentrations were varied at 0, 0.5, and 2
mM.
Crystal Structure and mutagenesis of TcAANAT0
In order to get a better structural understanding of TcAANAT0, we attempted to crystalize the apo
protein. However, crystallization efforts with the apo TcAANAT0 proved unsuccessful after
several attempts. We next attempted to co-crystallize TcAANAT0 in the presence of acetyl-CoA,
with the hope acetyl-CoA would sufficiently stabilize the protein to allow for crystal formation.
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The crystal structure of TcAANAT0 in complex with acetyl-CoA (Figure 3, PDB ID: 6V3T) was
determined at 2.84 Å resolution, and belongs to the C2 space group. The crystal has six copies of
TcAANAT0 in the asymmetric unit, with each copy bound to acetyl-CoA. The Fo-Fc electron
density for acetyl-CoA is well-defined with the exception of the carboxylate tail, where the density
is weaker than the rest of the compound. The adenine portion of acetyl-CoA forms a prominent
stacking interaction with Phe-166 and another acetyl-CoA present in an adjacent TcAANAT0
molecule at the crystal packing interface (Figure 3). Notably, no other AANAT structure has
shown such an interaction, highlighting the heterologous nature of iAANATs. Creation and
analysis of the F166A mutant illustrated the functional importance of this residue, as the F166A
mutant is catalytically inert enzyme (Table 3). This result is based on the lack of appreciable
activity above the background rate at any reasonable concentration of substrates. These stacking
interactions between adjacent acetyl-CoA molecules, and Phe-166, likely served to stablize the
lattice and helped to induce crystal formation. This possibility is further evidenced by the lack of
crystal formation when TcAANAT0 was incubated in the absence of acetyl-CoA.
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Table 3: Steady-state kinetic constants for TcAANAT0 site-directed mutants. Kinetic constants were measured in
triplicate as previously described for the wild-type enzyme
Mutant
Acetyl-CoA
KM (M)
25.0 ± 1.8
12.0 ± 2.4
8.7 ± 1.8
394 ± 22
N/A
Tryptamine
KM (M)
260 ± 29
751 ± 46
500 ± 68
1130 ± 75
N/A
kcat (s-1)
29 ± 0.6
1.1 ± 0.1
2.3 ± 0.1
29 ± 0.7
N/A
kcat/KM (M-1s-1)
% Effectiveness
Wild-Type
E25A
E25D
R134A
F166A
Mutant
(1.2 ± 0.1) × 106 100
(9.7 ± 2.1) × 104 3.8
(2.6 ± 0.5) × 105 22.1
(7.4 ± 0.4) × 104 6.3
N/A
0
kcat (s-1)
33 ± 0.8
1.40 ± 0.1
2.2 ± 0.1
12.8 ± 0.2
N/A
kcat/KM (M-1s-1)
% Effectiveness
Wild-Type
E25A
E25D
R134A
F166A
(9.2 ± 0.8) × 104 100
(1.8 ± 0.1) × 103 4.1
(4.5 ± 0.6) × 103 4.8
(1.1 ± 0.1) × 104 12.2
N/A
0
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A
B
C
D
Figure 3: Crystal Structure of TcAANAT0 in complex with acetyl-CoA shown at 2.84 Å resolution. (A)
Asymmetric unit demonstrating hexameric crystal packing. Monomer is shown in red and yellow (B) Monomer
of TcAANAT0 complexed with acetyl-CoA (C) TcAANAT0 active site in complex with acetyl-CoA. The unbiased
Fo-Fc electron density map is contoured at 3σ and shown in green. TcAANAT0 is colored in magenta, and acetyl-
CoA is colored in orange. Hydrogen bonds are shown as black dashed lines. (D) Pi-stacking arrangement of
acetyl-CoA and Phe-166 across two TcAANAT0 molecules at the crystal packing interface. TcAANAT0 is
colored in magenta and teal, and acetyl-CoA is colored in orange blue.
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Further examination of the binding of acetyl-CoA to TcAANAT0 demonstrates favorable
interactions with multiple active site residues (Figure 3). Specifically, the three phosphates in
acetyl-CoA form several potential hydrogen bonds with the TcAANAT0 backbone in the area from
Gly-135-Lys-140, as well as with the Lys-140 side chain. The pantothenic acid portion of acetyl-
CoA establishes extensive interactions with the active site. Noteably, Arg-134 forms a hydrogen
bond with acetyl-CoA, in addition to van der Waals interactions between the acetyl-CoA methyl
group and the Arg-134 side chain. Arg-134 aligns with Arg-153 of DmAANATA41, Arg-138 of
DmAANAT249, and Arg-138 of DmAANAT750, which have been shown to be important in
maintaining the structure of the acyl-CoA binding pocket (Figure 3). Production of the R134A
mutant of TcAANAT0 confirmed this, with a 94% and 88% drop in catalytic effeciency for acetyl-
CoA and tryptamine. A 15-fold increase in KM and preservation of kcat for acetyl-CoA is testament
to the structural rather than catalytic importance of Arg-134.
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Sequence alignment with other characterized iAANATs also indicated Glu-25 located in
close proximity to the amine binding pocket and the acetyl group of the bound acetyl-CoA holds
catalytic relevance in TcAANAT0 (Figure S5). This is due to its correlation to Glu-47 of
DmAANATA,41 Glu-26 of DmAANAT7,50 Glu-34 of DmAgmNAT51 and Glu-27 in Bm-
iAANAT3,40 which have all been shown to serve as a general base during catalysis.
Kinetic analysis of an E25A mutant of TcAANAT0 was in similar agreement, illustrated
by a 92% and 98% drop in catalytic effeciency for acetyl-CoA and tryptamine respectively.
Surprisingly, production of an E25D conserved mutant yielded similar results, with a 78% and
95% drop in catalytic effeciency recorded for acetyl-CoA and tryptamine, respectively. It is
interesting to note that shortening the length of the residue R-group by one methylene group
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significantly deters the ability of the enzyme to perform catalysis. Further structural analysis would
be needed to examine this interesting phenomenon in more detail.
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TcAANAT0 Chemical Mechanism
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To determine the chemical mechanism of TcAANAT0, the pH-dependence of binding and
catalysis was measured for acetyl-CoA, tryptamine, and 2,2-difluoro-2-phenethylamine (DFPE).
Rate profiles allowed the determination of likely catalytic or structurally important residues based
on their apparent pKa values (Figure S6). The best fits of the pH profile data to eqns. 10-12 yield
pKa values for ionizable groups involved in binding and/or catalysis (Table 4). An acidic pKa of
approximately 7.3 was attributed to a general base, which has classically been found in
iAANATs.40,41,49,51–54 The exact function of this general base has been argued, but it has been
proposed deprotonation of the amine occurs prior to formation of a tetrahedral intermediate.41 To
investigate this, rate profiles were generated for 2,2’-difluoro-2-phenethylamine (DFPE; predicted
pKa ≈ 6.7 ± 0.3 – calculated via SciFinder using Advanced Chemistry Development (ACD/Labs)
Software 11.02). The comparatively low pKa of this molecule allows us to directly test if amine
deprotonation is driven by the general base. The disappearance of the acidic limb when measuring
kcat against pH highlights that at pH levels of 6 to 8, where DFPE is still largely found in its
deprotonated form (when compared to tryptamine, pKa ≈ 10.2),55 the function of the general base
is negated.
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Table 4: Summary of pKa values measured from pH rate profiles
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Acidic Limb pKa values
kcat VS pH
Basic Limb pKa values
kcat VS pH
Acetyl CoA
Tryptamine
DFPE
9
Acetyl CoA
Tryptamine
DFPE
7.4 ± 0.1
9.6 ± 0.3
8.3 ± 0.2
8.7 ± 0.1
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7.1 ± 0.2
N/A
kcat / KM VS pH
Acetyl CoA
Tryptamine
DFPE
kcat / KM VS pH
Acetyl CoA
Tryptamine
DFPE
6.5 ± 0.2
7.9 ± 0.1
7.5 ± 0.2
8.7 ± 0.3
9.3 ± 0.2
8.6 ± 0.2
A second pKa of approximately 9.0 was also observed, with evidence indicating two
unresolvable species are involved. The first of these can likely be attributed to the thiol group of
the departing CoA-SH, which has a reported pKa ≈ 9.8.55 This pKa has also been attributed to a
general acid, which facilitates the release of the products. However, after careful examination of
the TcAANAT0 crystal structure, there is no obvious active site residue that might function as a
general acid during catalysis.
With this in mind, we propose the following chemical mechanism for TcAANAT0, with
acetyl-CoA and tryptamine acting as example substrates: Acetyl-CoA binding preceeds amine
binding. This subsequently leads to some undetermined structural movement, as implied by the
time-dependent inhibition of TcAANAT0 by CoA-S-acetyltryptamine. This allows for an
unidentified residue to move into position and polarize Glu-25 via hydrogen bonding, accounting
for the necessary increase in the apparent pKa of Glu-25. Nucleophilic attack at the carbonyl of the
thioester of acetyl-CoA then generates a zwitterionic tetrahedral intermediate. Subsequent
protonation of the CoA-SH moiety leads to intermediate breakdown. Upon product release, the
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polarizing ‘acidic’ residue moves out of hydrogen bond range of the base, thereby relaxing the pKa
of Glu-25. This results in its deprotonation and reformation of the enzyme active site. This
mechanism (Figure 4) follows, and builds upon, mechanisms proposed for other
iAANATs.41,49,51,56
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O
Acetyl-CoA
TcAANAT0
Tryptamine
TcAANAT0:Acetyl-CoA
S CoA
H
H
N
N
H2
B
AH
H
H
N
O
N
O
N
HN
H
S CoA
H
+
B
BH
CoA SH
AH
Figure 4: Proposed chemical mechanism for T. castaneum AANAT0. The general base was identified as Glu-25.
The polarizing ‘acid’ only moves into proximity of the base once the amine binds, and thus remains elusive.
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Conclusion
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The knowledge gained from such an intrinsic understanding of the mechanistic and structural
relationship of iAANATs is invaluable in their development as insecticide targets. The
fundamental differences, such as binding order, chemical mechanism, and substrate specificity,
which make these enzymes so appealing as insecticide targets, is also what warrants their
individual examination. Fragment-based drug discovery in the pursuit of AANAT-targeted
inhibitors with insect specificity is the natural progression of this work, and such investigations
are in progress. These investigations are based on interactions noted in the acyl-CoA binding
pocket of insect AANAT crystal structures. By beginning with an amine base fragment, such as
tyramine, and extending into the acyl-CoA binding pocket, costly CoA reagents are negated. This
results in a panel of inhibitors which are readily synthesizable, tight binding, and economic. This
promising work is ongoing, with efforts currently advancing from in silico to in vitro.
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Methods
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Determination of enzyme activity
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4,4’-Dithiodipyridine (DTDP) was used to determine the activity of TcAANAT0 at 324 nm (molar
absorptivity = 19,800 M-1cm-1)57 by measuring the formation of CoA-SH product. Each 750 L
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assay consisted of 300 mM Tris, pH 8.0, 375 M DTDP, and the desired concentration of acyl-
CoA and amine substrates. Amine and acyl-CoA kinetic constants were measured by holding the
concentration of acetyl-CoA and tryptamine at fixed, saturated levels (250 M and 5 mM)
respectively. Measurements were made in triplicate using a Cary 300 Bio UV−visible
spectrophotometer at 30 °C. Steady-state kinetic parameters were evaluated by fitting initial
velocity measurements to Equation 1 using Enzyme Kinetic Wizard (SigmaPlot 12.0).
Equation 1
푉푚푎푥[푆]
푉표 =
퐾푀 + [푆]
Determination of kinetic mechanism
To fully define the kinetic mechanism of TcAANAT0, a combination of initial velocity double-
reciprocal analysis and dead-end inhibition was utilized. Firstly, initial velocities were measured
by varying the concentration of one substrate while holding the other substrate at constant, but
different concentrations. Measurements were made at acetyl-CoA concentrations of 10, 20, 37,
and 100 M, and tryptamine concentrations of 50, 75, 125, 360, and 720 M. The resulting data
were fit to Equation 2 for an ordered bi-bi mechanism using Visual Enzymics 2010 (IGOR Pro
6.34 A)
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Equation 2
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푉푚푎푥[퐴][퐵]
퐾푖,푎퐾푀,푏 + 퐾푀,푎[퐵] + 퐾푀,푏[퐴] + [퐴][퐵]
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To further define the kinetic mechanism, tryptophol and desulfo-CoA were utilized as
dead-end inhibitors for tryptamine and desulfo-CoA respectively. Initial velocity patterns were
obtained by varying the concentration of one substrate, while holding the concentration of the
second at approximately the KM. This was then repeated twice in the presence of fixed
concentrations of inhibitor. To test inhibition of the amine binding site, tryptophol was used as the
inhibitor at 2.0 and 4.0 mM. To test inhibition of the acyl-CoA binding site, desulfo-CoA was
introduced as the inhibitor at 500 and 1000 M. Measurements were made at varying tryptamine
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and acetyl CoA concentrations of 50, 75, 100, 150, 300 and 500 M, and 10, 20, 30, 50, 80 and
100 M respectively. To measure the steady-state inhibition of CoA-S-acetyltryptamine,
measurements were made at varying tryptamine and acetyl CoA concentrations of 50, 75, 100,
150, 300 and 500 M, and 20, 30, 50, 80, 100 and 120 M respectively and at 0, 100 and 250 M
CoA-S-acetyltryptamine. The data were then fit to Equation 3, Equation 4, and Equation 5 using
Enzyme Kinetic Wizard (SigmaPlot 12.0) to differentiate between competitive, non-competitive,
and uncompetitive inhibition.
Equation 3
푉푚푎푥[푆]
퐼
퐾푀 1 +
+ [푆]
(
)
퐾푖푠
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Equation 4
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푉푚푎푥[푆]
퐼
1
퐾푀 1 +
+ [푆] 1 +
(
)
(
)
퐾푖푠
퐾푖푖
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Equation 5
푉푚푎푥[푆]
1
퐾푀 + [푆] 1 +
(
)
퐾푖푖
TcAANAT0 Mutagenesis
To investigate the importance of individual residues in substrate binding and catalysis, alanine
scanning was performed on several residues which have classically shown to be important in
AANAT activity, or are shown via crystal structure to be of interest. The mutants made were:
E25A, E25D, R134A, and F166A. Primers for these mutants were designed using the Agilent
QuikChange Primer Design tool, and the mutants were synthesized using an Agilent QuikChange
Ligntning kit following the recommended procedure. Following transformation, all mutant DNA
was sequenced to confirm successful mutation. The mutant enzymes were cultured, purified and
kinetically analyzed following the same procedures as described above.
Supporting Information
The Supporting Information is available free of charge via the internet on the ACS Publications
website at http://pubs.acs.org
Cloning of T. castaneum AANAT0, product characterization via mass spectrometry, determination
of acetyl-CoA binding constants via isothermal titration calorimetry, characterization of
bisubstrate analog CoA-S-acetyltryptamine time-dependent inhibition, insect AANAT sequence
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alignment, determination of pH-dependence, and crystallography including x-ray data collection
and refinement.
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Accession Codes
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Atomic coordinates and experimental details for the crystal structure of TcAANAT0, 6V3T, will
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be released upon publication.
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Acknowledgements
This work has been supported, in part, by grants from the University of South Florida (a Creative
Scholarship grant), the Shirley W. and William L. Griffin Charitable Foundation, the National
Institute of Drug Abuse at the National Institutes of Health (R03-DA034323) and the National
Institute of General Medical Science of the National Institutes of Health (R15-GM107864) to D.J.
Merkler and the National Institute of General Medical Sciences (R01-GM115854) to I. Gelis. This
work has also received support from the Mass Spectrometry and Peptide Facility, Department of
Chemistry, University of South Florida.We also thank the SBC beamline scientists for assistance
with data collection. This research used resources of the Advanced Photon Source, a U.S.
Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of
Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Results
shown in this report are derived from work performed at Argonne National Laboratory, Structural
Biology Center (SBC) at the Advanced Photon Source. SBC-CAT is operated by UChicago
Argonne, LLC, for the U.S. DOE, Office of Biological and Environmental Research under
Contract No. DE-AC02-06CH11357.
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