Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases

Article

pubs.acs.org/biochemistry

Mechanistic and Structural Analysis of Drosophila melanogaster

Arylalkylamine N‑Acetyltransferases

Daniel R. Dempsey,^†Kristen A. Jeﬀries,^†Jason D. Bond,^†Anne-Marie Carpenter,^†,⊥

Santiago Rodriguez-Ospina,^†Leonid Breydo,^‡,§K. Kenneth Caswell,^∥and David J. Merkler*^,†

^†Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States

§

^‡Department of Molecular Medicine and Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South

Florida, Tampa, Florida 33612, United States

^∥College of Arts and Sciences, University of South Florida Sarasota-Manatee, Sarasota, Florida 34243, United States

S

* Supporting Information

ABSTRACT: Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and

other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a

critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two

AANAT variants (AANATA and AANATB) have been identiﬁed in D. melanogaster, in which AANATA diﬀers from AANATB

by the truncation of 35 amino acids from the N-terminus. We have expressed and puriﬁed both D. melanogaster AANAT variants

(AANATA and AANATB) in Escherichia coli and used the puriﬁed enzymes to demonstrate that this N-terminal truncation does

not aﬀect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identiﬁed

an ordered sequential mechanism, with acetyl-CoA binding ﬁrst, followed by tyramine. We used a combination of pH−activity

proﬁling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to

an assignment of Glu-47 as the general base in catalysis with an apparent pK_aof 7.0. Using the data generated for the kinetic

mechanism, structure−function relationships, pH−rate proﬁles, and site-directed mutagenesis, we propose a chemical mechanism

for AANATA.

hydroxylated to generate octopamine, a reaction that is

iogenic amines are important neuroactive amines found in

B_{both vertebrates and invertebrates functioning as neuro-}catalyzed by tyramine β-hydroxylase.⁸⁻¹⁰One proposed

inactivation reaction for the arylalkylamines is N-acetylation

as catalyzed by arylalkylamine N-acetyltransferase (AANAT):

acetyl-CoA + arylalkylamine → N-acetylarylalkylamine + CoA-

SH.^11,12In addition to arylalkylamine inactivation, acetyl-CoA-

dependent N-acetylation is involved in cuticle sclerotiza-

tion¹³⁻¹⁶and melatonin biosynthesis.^17,18

AANAT (EC 2.3.1.87), also known as serotonin N-

acetyltransferase and dopamine N-acetyltransferase, has been

identiﬁed in Drosophila melanogaster¹¹and is a member of the

GCN5-related N-acetyltransferase (GNAT) superfamily.^19,20

The identiﬁcation of N-acetyltransferase activity for arylalkyl-

amines in D. melanogaster was ﬁrst shown in 1972 by Dewhurst

et al.,¹¹followed by the initial characterization of AANAT in

1977 by Maranda and Hodgetts.²¹In 1998, Brodbeck et al.²²

identiﬁed two biologically relevant variants, variant A

(AANATA) and variant B (AANATB), which diﬀer by 35

transmitters, neuromodulators, or neurohormones via their

binding to speciﬁc receptors. In insects, the biogenic arylalkyl-

amines are dopamine, tyramine, serotonin, and octopamine,

which function primarily as neurotransmitters.¹The pathways

for biosynthesis and degradation have important roles in the

function of the arylalkylamines as a balance between production

and clearance is necessary to maintain the appropriate cellular

concentrations of the amines. Dysfunction in either of these

opposing pathways would lead to improper cellular levels of the

arylalkylamines, leading to errors in the processes regulated by

them.²

The biogenic arylalkylamines are all derived in vivo from the

cognate aromatic amino acid (tyrosine or tryptophan)

precursor. The precursor amino acid is initially hydroxylated

by an aromatic amino acid hydroxylase (tyrosine hydroxylase or

tryptophan hydroxylase)^3,4and then decarboxylated by

aromatic ^L-amino acid decarboxylase (3,4-dihydroxylphenylala-

nine decarboxylase)⁵to generate either dopamine or serotonin,

respectively. Tyramine is derived from the decarboxylation of

tyrosine by tyrosine decarboxylase,^6,7which can then be β-

Received: May 19, 2014

Revised: November 17, 2014

A

dx.doi.org/10.1021/bi5006078 | Biochemistry XXXX, XXX, XXX−XXX

Biochemistry

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amino acids found only at the N-terminus of the larger

AANATB. Diﬀerential transcription of a single AANAT gene

leads to the two diﬀerent AANAT variants.²²These variants are

expressed in diﬀerent tissues and at diﬀerent life stages, with

AANATA being found in the brain, ventral nerve cord, and

midgut during late stage embryogenesis and in adults.

AANATB is less abundant and is found in the brain only

during late pupal stages and in adults, as well.²²AANAT

enzymes have been characterized from many organisms and are

known to catalyze the rate-limiting penultimate step in the

formation of melatonin.^20,23−25Melatonin is a hormone that is

produced in a diurnal cycle²⁴(higher levels are found at night)

and is suggested to regulate the life span of D. melanogaster.¹⁸

Eight putative AANAT-like enzymes (AANATL) have been

identiﬁed in D. melanogaster²⁶and are proposed to play a role

in the biosynthesis of fatty acid amides.^27,28Herein, we report

on the cloning, expression, puriﬁcation, and characterization of

D. melanogaster AANATA and AANATB from recombinant

Escherichia coli. Our data show that AANATA and AANATB

are catalytically similar with respect to substrate speciﬁcities and

measured kinetic constants (K_m,appand k_cat,app). More detailed

studies of AANATA were performed to deﬁne the kinetic

mechanism and to elaborate a chemical mechanism that is

consistent with both the pH−rate proﬁles and data from a set

of site-directed mutant enzymes.

Luria broth (LB) supplemented with 40 μg/mL kanamycin at

37 °C. The plasmids were puriﬁed using the Promega Wizard

Plus SV Minipreps DNA puriﬁcation kit and sequenced by

Euroﬁns MWG operon. In separate experiments, the AANATA

pET-28a or AANATB pET-28a vector was then transformed

into E. coli BL21(DE3) cells for expression of AANATA or

AANATB, respectively.

Expression and Puriﬁcation of AANATA and AANATB.

The E. coli BL21(DE3) cells harboring either the AANATA

pET-28a or AANATB pET-28a expression vector were cultured

in LB supplemented with 40 μg/mL kanamycin at 37 °C. The

cell cultures were induced at an OD₆₀₀of 0.6 with 1 mM

isopropyl β-^D-1-thiogalactopyranoside for 4 h at 37 °C. The

ﬁnal cultures were then harvested by centrifugation at 5000g for

10 min at 4 °C, and the pellet was collected.

The pellet was then resuspended in 20 mM Tris (pH 7.9),

500 mM NaCl, and 5 mM imidazole; the cells were lysed by

sonication, and the cellular debris was removed by

centrifugation (10000g for 15 min at 4 °C). The supernatant

was loaded onto a column packed with 6 mL of ProBond

nickel-chelating resin. The column was ﬁrst washed with 10

column volumes of 20 mM Tris-HCl (pH 7.9), 500 mM NaCl,

and 5 mM imidazole and then with 10 column volumes of 20

mM Tris-HCl (pH 7.9), 500 mM NaCl, and 60 mM imidazole,

and AANATA or AANATB eluted in 1 mL fractions of 20 mM

Tris-HCl (pH 7.9), 500 mM NaCl, and 500 mM imidazole.

Purity was assessed by 10% sodium dodecyl sulfate−

polyacrylamide gel electrophoresis (SDS−PAGE) and visual-

ized using Coomassie stain. The protein concentration was

determined using the Bradford dye binding assay.

Production of Site-Directed Mutants. Each site-directed

AANATA mutant was generated by the overlap extension

method²⁹using PfuUltra High-Fidelity DNA polymerase under

the following PCR conditions: initial denaturing step of 95 °C

for 2 min, then 30 cycles (95 °C for 30 s, 60 °C for 30 s, and 72

°C for 1 min), and then a ﬁnal extension step of 72 °C for 10

min. Primers for each mutant (Table S1 of the Supporting

Information) were designed with the Agilent QuickChange

Primer Design tool. The AANATA mutant PCR products were

then inserted into a pET-28a(+) vector using the NdeI and

XhoI restriction enzymes. The AANATA mutant pET-28a

vectors were transformed into E. coli XL10 competent cells and

cultured in LB supplemented with 40 μg/mL kanamycin at 37

°C. The plasmids were then puriﬁed using the Promega Wizard

Plus SV Minipreps DNA puriﬁcation kit and sequenced by

Euroﬁns MWG operon. Individual mutant AANATA proteins

were expressed and puriﬁed as described previously for the

wild-type enzyme.

EXPERIMENTAL PROCEDURES

■

Materials. The Ambion RETROscript Kit, ProBond nickel-

chelating resin, and MicroPoly(A) Purist were purchased from

Invitrogen. NdeI, XhoI, Antarctic Phosphatase, and T4 DNA

ligase were purchased from New England Biolabs. BL21(DE3)

E. coli cells, XL10 E. coli cells, and the pET-28a(+) vector were

purchased from Novagen. Kanamycin monosulfate and

isopropyl β-^D-1-thiogalactopyranoside were purchased from

Gold Biotechnology. Oligonucleotides were purchased from

Euroﬁns MWG Operon, and PfuUltra High-Fidelity DNA

polymerase was purchased from Agilent. Benzoyl-CoA, acetyl-

CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-

CoA, oleoyl-CoA, and N-acetylserotonin were purchased from

Sigma-Aldrich. All other reagents were of the highest quality

available from either Sigma-Aldrich or Fisher Scientiﬁc.

Cloning of D. melanogaster AANATA and AANATB. A

cDNA library was generated from D. melanogaster heads, using

the Ambion RETROscript Kit and MicroPoly(A) Purist kits.

AANATA (NCBI reference sequence NM_079115.2) and

AANATB (NCBI reference sequence NM_206212.1) were

ampliﬁed from the D. melanogaster head cDNA library using the

following primers: 5′ GAC TCA TAT GAT GGA GGA CGC

ATT GAC C 3′ (forward) and 5′ ATC CCT CGA GCT ACA

GCT TGG TCT GCG C 3′ (reverse); 5′ GCT ACA TAT

GAT GGA AGT GCA GAA GCT (forward) and 3′ ATC CCT

CGA GCT ACA GCT TGG TCT GCG C (reverse),

respectively. Polymerase chain reaction (PCR) was performed

using PfuUltra High-Fidelity DNA polymerase, using the

following set of conditions: initial denaturing step of 95 °C

for 2 min, then 30 cycles (95 °C for 30 s, 60 °C for 30 s, and 72

°C for 1 min), and then a ﬁnal extension step of 72 °C for 10

min. The AANATA product or the AANATB PCR product was

inserted into a pET-28a(+) vector using the NdeI and XhoI

restriction enzymes, yielding an expression vector for each

enzyme, AANATA pET-28a or AANATB pET-28a, respectively.

The AANATA pET-28a or AANATB pET-28a vector was then

transformed into E. coli XL10 competent cells and cultured in

Measurement of Enzyme Activity. AANATA and

AANATB activity was analyzed using Ellman’s reagent³⁰by

measuring the release of coenzyme A at 412 nm in 300 mM

Tris-HCl (pH 8.0), 150 μM DTNB, and the desired

concentrations of the amino donor substrate and acyl-CoA

substrate. Initial velocities of CoA-SH release were measured

using a Cary 300 Bio UV−visible spectrophotometer, and the

resulting initial velocity kinetic data were ﬁt to the desired

equation using SigmaPlot 12.0. Steady-state kinetic constants

were obtained by a ﬁt to eq 1

V

_max[S]

v =

o

K_m+ [S]

(1)

B

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where v_ois the initial velocity, [S] is the substrate

concentration, V_maxis the maximal velocity, and K_mis the

Michaelis constant.

V

_max[S]

v =

o

[I]

K_i

K_m+ [S] 1 +

(

)

(7)

Apparent kinetic constants for each arylalkylamine substrate

were determined by holding acetyl-CoA at a ﬁxed saturating

concentration, whereas those for each acyl-CoA substrate were

determined by holding tyramine at a ﬁxed saturating

concentration at 22 °C. Assays were performed in triplicate,

and the uncertainties for the (k_cat/K_m)_appand relative (k_cat/

K_m)_appvalues were deﬁned by using eq 2, where σ is the

standard error.³¹

where v_ois the initial velocity, V_maxis the maximal velocity, [S]

is the substrate concentration, K_mis the Michaelis constant, [I]

is the inhibitor concentration, and K_iis the inhibition constant.

The assays were performed in triplicate.

Rate versus pH Dependence. The pH dependence of the

steady-state kinetic constants was determined for both acetyl-

CoA and tyramine as varied substrates while holding the other

substrate at a ﬁxed concentration. Kinetic constants were

determined at 0.5 pH intervals from pH 6.0 to 9.5, using the

following buﬀers: MES (pH 6.0−7.0), Tris (pH 7.0−9.0), and

2-amino-2-methyl-1-propanol (AMeP) (pH 9.0−9.5). The

resulting data were ﬁt to eq 8 [log(k_cat/K_{m‑acetyl‑CoA}) or

log(k_cat/K_m‑tyramine)] and eq 9 (log k_cat)

2

⎛ ⎞

⎛

⎞

σ_y

y

σ

x

⎛

⎜

⎞

⎟

x

y

x

σ

=

+

⎜ ⎟

⎜

⎝

⎟

⎠

⎝

⎠

y

⎝ ⎠

(2)

Kinetic Mechanism and Inhibitor Analysis. Initial

velocity patterns for acetyl-CoA and tyramine were produced

by varying the concentration of one substrate while holding the

concentration of the other substrate at a ﬁxed concentration

and ﬁtting the data to eq 3 for an ordered bi-bi mechanism

using Igor Pro 6.34A

log(k_cat/K_m) = log[c/(1 + 10^{pK −pH})]

a

(8)

log(k_cat) = log[c/(1 + 10^{pK −pH}+ 10^pH−pK)]

b

a

(9)

where c is the pH-independent plateau using Igor Pro 6.34A.

Intrinsic Fluorescence Measurements for the Deter-

mination of the Coenzyme A Dissociation Constant.

Fluorescence spectra were generated with a JASCO FP-8300

spectroﬂuorometer equipped with a circulating water bath

maintained at 22 °C. Emission spectra (excitation at 280 nm,

emission at 290−300 nm) were measured in a 0.4 cm path

length cell containing 400 μL of 300 mM Tris-HCl (pH 8.0),

varying concentrations of coenzyme A, and a ﬁxed AANATA

enzyme concentration (0.08 mg/mL for the wild type, 0.1 mg/

mL for R153A). Fluorescence emission spectra were acquired

in triplicate, with a scan speed of 50 nm/min, an excitation

bandwidth of −5 nm, and an emission bandwidth of −2.5 nm.

The K_dfor coenzyme A was determined by ﬁtting the data to

eq 10 with SigmaPlot 12.0

V

_max[A][B]

v =

K_iaK_b+ K_a[B] + K_b[A] + [A][B]

(3)

where K_iais the dissociation constant for substrate A (acetyl-

CoA), K_bis the K_mfor substrate B (tyramine), and K_ais the K_m

for substrate A (acetyl-CoA). The two experimental sets were

generated by holding the tyramine concentration constant (5,

10, 25, and 50 μM) and varying the concentration of acetyl-

CoA, whereas the second set involved holding the concen-

tration of acetyl-CoA constant (20, 40, 60, and 100 μM) and

varying the concentration of tyramine.

The IC₅₀values for long-chain acyl-CoAs and tyrosol were

determined at the K_mconcentration for the appropriate

substrate, tyramine at 12 μM or acetyl-CoA at 39 μM, while

varying the concentration of the respective inhibitor. The IC₅₀

value was determined by a ﬁt of the resulting data to eq 4 using

SigmaPlot 12.0

ΔF_max[L]

ΔF =

K_d+ [L]

(10)

v_i

1

where ΔF is the change in intrinsic ﬂuorescence, ΔF_maxis the

maximal change in ﬂuorescence at inﬁnite ligand concentration,

K_dis the ligand dissociation constant, and [L] is the ligand

concentration.

=

[I]

IC₅₀

v

1 +

o

(4)

where v_ois the initial velocity without inhibitor, v_iis the initial

velocity at diﬀerent inhibitor concentrations, and [I] is the

inhibitor concentration. The assays were performed in

triplicate.

Dead-end inhibitor analysis was conducted for both oleoyl-

CoA and tyrosol. The inhibition patterns were determined by

holding one substrate (acetyl-CoA or tyramine) at a ﬁxed

concentration and varying the concentration of the other

substrate, with each set conducted at a diﬀerent ﬁxed

concentration of the inhibitor. The initial velocities from

these inhibitor experiments were ﬁt to eqs 5−7 for competitive,

noncompetitive, and uncompetitive inhibition, respectively

Product Characterization. Product characterization was

performed using a Phenomenex Kinetex 2.6 μm C₁₈100 Å (50

mm × 2.1 mm) reverse phase column coupled with an Agilent

6540 liquid chromatography/quadrupole time-of-ﬂight mass

spectrometer (LC/QTOF-MS) in positive ion mode. An

enzyme reaction mixture comprised of 300 mM Tris-HCl

(pH 8.0), 500 μM acetyl-CoA, 1 mM serotonin, and 5 μg

AANATA in a ﬁnal volume of 750 μL was incubated for 30 min

at room temperature. Then AANATA was removed from the

reaction mixture by centrifugation using a Millipore 10 kDa

ﬁlter. The resulting sample was injected on the LC/QTOF-MS,

and the retention time and high-resolution mass were

compared with those of a commercial standard of N-

acetylserotonin. Conditions for LC/QTOF-MS analysis are

described in ref 27.

V

_max[S]

v =

o

[I]

K_i

K_m1 +

+ [S]

(

)

(5)

(6)

V

_max[S]

RESULTS

■

v =

o

[I]

K_i

[I]

K_i

Overexpression and Puriﬁcation of AANATA and

AANATB. AANATA and AANATB were cloned from a

K_m1 +

+ [S] 1 +

(

)

(

)

C

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Table 1. Steady-State Kinetic Constants for AANATA and AANATB with Diﬀerent Acyl-CoAs

a

substrate

K_m,app(μM)

k_cat,app(s⁻¹

AANATA

0.37 0.03

16

)

(k_cat/K_m)_app(M⁻¹s⁻¹

)

relative (k_cat/K_m)_app

bc

,

benzoyl-CoA

acetyl-CoA

65 13

39 12

(5.7 1.2) × 10³

(4.1 1.3) × 10⁵

(2.8 0.2) × 10⁵

(6.8 0.9) × 10⁴

(1.4 0.2) × 10⁴

(1.7 0.5) × 10²

1.0 0.3

72 27

49 11

1

butyryl-CoA

hexanoyl-CoA

octanoyl-CoA

decanoyl-CoA

36

23

18

2

3

9.9 0.4

1.6 0.1

12

3

0.26 0.01

0.04 0.01

2.5 0.6

220 60

0.04 0.01

cd

,

AANATB

benzoyl-CoA

acetyl-CoA

80

64

19

23

6

9

3

6

0.12 0.04

8.3 0.4

(1.6 0.5) × 10³

(1.3 0.2) × 10⁵

(2.3 0.3) × 10⁵

(3.8 1.0) × 10⁴

1.0 0.4

81 31

150 55

25 10

butyryl-CoA

hexanoyl-CoA

4.4 0.2

0.88 0.04

a

b

Reaction condition: 300 mM Tris-HCl (pH 8.0), 150 μM DTNB, 1.0 mM tyramine, and varying acyl-CoA concentrations. Relative (k_cat/K_m)_app

c

values for AANATA amino acceptor substrates were indexed to benzoyl-CoA. Kinetic constants are reported with the standard error (n = 3).

d

Relative (k_cat/K_m)_appvalues for AANATB amino acceptor substrates were indexed to benzoyl-CoA.

cDNA library generated from the D. melanogaster head (Figure

S1 of the Supporting Information). Both genes were inserted

into a pET28a(+) vector that encodes an N-terminal His₆tag.

Both AANATA and AANATB were puriﬁed using ProBond

nickel-chelating resin to homogeneity, yielding 21 mg of

AANATA or 3 mg of AANATB per liter of culture. Purity was

assayed by SDS−PAGE (Figure S2 of the Supporting

Information) and determined to be >95%.

ferases.^32,33The (K_m,app

)

is similar to the values we

benzoyl‑CoA

measured for short-chain acyl-CoA thioesters, but the k_cat,appis

signiﬁcantly lower, such (k_cat/K_m)_{app,benzoyl‑CoA}is ∼1.5% of (k_cat/

K_m)_{app,acetyl‑CoA}

.

Characterization of the Amino Donor Substrates.

Amino donors are deﬁned herein as compounds that contain a

free amino moiety. The substrate speciﬁcity of AANATA was

evaluated using a set of arylalkylamine amino donors; these

data show that tyramine is the amino donor with the highest

(k_cat/K_m)_appvalue at saturating acetyl-CoA (Table 3). The

kinetic constants for tyramine, with both AANATA and

AANATB, were very similar at saturating acetyl-CoA

Characterization of the Amino Acceptor Substrates.

Amino acceptors are deﬁned herein as any acyl-CoA or aryl-

CoA substrate. Our data for AANATA and AANATB (Table 1)

show that the amino acceptor speciﬁcities are approximately the

same for both enzymes. Both catalyze the formation of N-

acyltyramines, utilizing a set of straight-chain saturated acyl-

CoAs with little variation in the K_m,appvalues from acetyl-CoA

to hexanoyl-CoA for the two enzymes. A two-carbon increase

in acyl-chain length to decanoyl-CoA has a dramatic and

negative eﬀect on catalysis. The K_m,appis ∼10-fold higher and

the k_cat,app∼10-fold lower than the values for octanoyl-CoA,

such that the (k_cat/K_m)_{app,decanoyl‑CoA}is ∼1% of the (k_cat/

K_m)_{app,octanoyl‑CoA}and only ∼0.04% of the (k_cat/K_m)_{app,acetyl‑CoA}

(Table 1). Acyl-CoA thioesters possessing an acyl chain of ≥12

carbon atoms were not substrates for AANATA. Although

lauroyl-CoA and the longer-chain acyl-CoA thioesters were not

AANATA substrates, all were inhibitors of the enzyme with

IC₅₀values of ≤1.1 μM. We ﬁnd a slight increase in the

apparent binding aﬃnity as the acyl-chain length increases, with

the IC₅₀values decreasing from 1.1 μM for lauroyl-CoA to 0.4

μM for oleoyl-CoA (Table 2).

concentrations [for AANATB, (K_m,app

)

= 20

3 μM

tyramine

and (k_cat,app

)

= 16 1 s⁻¹compared with the AANATA

tyramine

data in Table 3].

A group of other arylalkylamines was evaluated as AANATA

amino donor substrates to better understand the general

structural features that aﬀect binding and catalysis (Table 3).

The (k_cat/K_m)_appvalues ranged over 2−3 orders of magnitude

for the amino donors we included in our study, with tyramine

as the best amino donor with the highest (k_cat/K_m)_appvalue and

3,4-dimethoxyphenethylamine as the worst amino donor

substrate with the lowest (k_cat/K_m)_appvalue. Tyramine and

tryptamine exhibit relatively high (k_cat/K_m)_appvalues (Table 3);

therefore, modiﬁcation at the α-position was explored to

determine if their cognate amino acids could serve as substrates.

No activity was observed above the baseline rate of acetyl-CoA

hydrolysis for tyrosine, tyrosine methyl ester, or tryptophan.

Next, we assessed tyrosine, tyrosine methyl ester, and

tryptophan as AANATA inhibitors at a concentration of 1.0

mM, with the acetyl-CoA (39 μM) and tyramine (12 μM)

concentrations ﬁxed at their respective K_m,appvalues. No

inhibition was observed for these three compounds.

The amino donor substrate speciﬁcity for AANATA was

further evaluated to determine if non-arylalkylamines could

serve as substrates. The decarboxylated amino acids, histamine

(histidine) and ethanolamine (serine), were not AANATA

substrates. Isoniazid, a ﬁrst-line drug for the treatment of

tuberculosis,³⁴⁻³⁶was evaluated for AANATA binding. There

was no observed decrease in the rate of reaction in the presence

of 1.0 mM isoniazid.

Benzoyl-CoA was also included in our analysis of amino

acceptor substrates to facilitate the comparison of our substrate

speciﬁcity data with those published for other N-acyltrans-

Table 2. AANATA IC₅₀Values for Long-Chain Acyl-CoAs

a

inhibitor

carbon skeleton

IC₅₀(nM)

380

oleoyl-CoA

18:1 (Δ⁹)

16:1 (Δ⁹)

14:0

5

palmitoleoyl-CoA

myristoyl-CoA

lauroyl-CoA

430 40

580 70

12:0

1100 130

a

Reaction was ﬁxed at the K_m,appvalues for both acetyl-CoA (39 μM)

Product Characterization. The product formed from the

AANATA reaction using acetyl-CoA and serotonin as

substrates was analyzed by LC/QTOF-MS in positive ion

and tyramine (12 μM) while varying the concentration of the long-

chain acyl-CoA inhibitor. Kinetic constants are reported with the

standard error (n = 3).

D

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Table 3. AANATA Steady-State Kinetic Constants for Diﬀerent Arylalkylamines

a

b

Reaction condition: 300 mM Tris-HCl pH 8.0, 150 μM DTNB, 500 μM acetyl-CoA, and varying concentration of arylalkylamine. Kinetic

constants are reported standard error (n = 3).

mode. Identiﬁcation of the product formed from this reaction

was compared with the commercial standard, N-acetylseroto-

nin. The AANATA product, N-acetylserotonin, was successfully

identiﬁed by comparison of both the retention time on a

Kinetex 2.6 μm C₁₈100 Å (50 mm × 2.1 mm) reverse phase

column and the [M + H]⁺(m/z) high-resolution mass

spectroscopy peak with those of the commercial standard

(Table S2 of the Supporting Information).

Kinetic Mechanism and Inhibitor Analysis. Initial

velocity plots were generated using tyramine and acetyl-CoA,

by varying the concentration of one substrate while holding the

other substrate at a ﬁxed saturated concentration. The resulting

double-reciprocal plots (Figure 1) reveal a pattern of

intersecting lines, suggesting a sequential kinetic mechanism

in which both substrates must be bound before catalysis can

occur. Inhibition studies were used to diﬀerentiate between a

random and an ordered sequential mechanism for AANATA,

using oleoyl-CoA and tyrosol as the dead-end inhibitors.

Oleoyl-CoA is an analogue of acetyl-CoA, whereas tyrosol is an

analogue of tyramine; neither is a substrate for AANATA.

Oleoyl-CoA is a competitive inhibitor (Figure 2) versus acetyl-

CoA and a pure noncompetitive inhibitor versus tyramine, with

inhibition constants of 78

8 nM and 150

9 nM,

respectively. Tyrosol is uncompetitive versus acetyl-CoA and

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Figure 1. Double-reciprocal plot of initial velocities for acetyl-CoA and tyramine. (A) Velocities measured at a ﬁxed concentration of tyramine: 50

●

○

▼

△

●

○

μM ( ), 25 μM ( ), 10 μM ( ), and 5 μM ( ). (B) Velocities measured at a ﬁxed concentration of acetyl-CoA: 100 μM ( ), 60 μM ( ), 40 μM

(

▼

△

), and 20 μM ( ).

Figure 2. Dead-end inhibition analysis of AANATA. (A) Velocities measured at a ﬁxed concentration of tyramine (12 μM), varying the

●

○

▼

concentration of acetyl-CoA, and varying the concentration of the inhibitor, oleoyl-CoA: 0 nM ( ), 200 nM ( ), and 375 nM ( ) (K_i= 78

8

nM). (B) Velocities measured at a ﬁxed concentration of acetyl-CoA (39 μM), varying the concentration of tyramine, and varying the concentration

●

○

▼

of the inhibitor, oleoyl-CoA: 0 nM ( ), 200 nM ( ), and 375 nM ( ) (K_i= 150 7 nM). (C) Velocities measured at a ﬁxed concentration of

●

○

tyramine (12 μM), varying the concentration of acetyl-CoA, and varying the concentration of the inhibitor, tyrosol: 0 μM ( ), 100 μM ( ), and 580

μM ( ) (K_i= 260 10 μM). (D) Velocities measured at a ﬁxed concentration of acetyl-CoA (39 μM), varying the concentration of tyramine, and

varying the concentration of the inhibitor, tyrosol: 0 μM ( ), 100 μM ( ), and 580 μM ( ) (K_i= 302 34 μM).

▼

●

○

▼

competitive versus tyramine, with inhibition constants of 260

10 μM and 302 34 μM, respectively.

pH Dependence of the Initial Rates. We studied the pH

dependence on the k_cat,appand (k_cat/K_m)_appfor both acetyl-CoA

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Figure 3. AANATA pH−rate proﬁle: (A) k_{cat,app‑acetyl‑CoA}, (B) k_{cat,app‑tyramine}, (C) (k_cat/K_m)_{app‑acetyl‑CoA}, and (D) (k_cat/K_m)_{app‑tyramine}

.

Figure 4. Crystal structure of D. melanogaster AANATA with bound acetyl-CoA identifying the possible general base residues. Stick model showing

the proximity of Glu-47, His-178, and Asp-142 to acetyl-CoA. Red spheres represent water molecules.

and tyramine. A rising pH proﬁle was observed for both (k_cat/

K_m)_{app‑acetyl‑CoA}(Figure 3C) and (k_cat/K_m)_{app‑tyramine}(Figure

3D). The data for both are best ﬁt using a single pK_a,appvalue,

yielding a pK_a,appvalue of 7.1 0.2 for the (k_cat/K_m)_{app‑acetyl‑CoA}

proﬁle and a pK_a,appvalue of 7.0 0.3 for the (k_cat/K_m)_{app‑tyramine}

proﬁle. The k_cat,appproﬁle is bell-shaped (Figure 3A,B) for

acetyl-CoA and tyramine, yielding two ionizable groups with

the same apparent pK_avalues, consisting of an apparent pK_aof

7.0 0.1 and an apparent pK_aof 9.8 0.2.

pH−rate proﬁle data provide the apparent pK_avalues for the

entities involved in catalysis but do not provide deﬁnitive

information concerning the identity of these chemical species.

Identiﬁcation of these species is critical for a more complete

understanding of the catalytic mechanism. One method we

used to pinpoint residues important for catalysis was the

alignment of the primary sequences of known AANAT-like

(AANATL) enzymes from D. melanogaster (Figure S3 of the

Supporting Information).²⁶

Site-Directed Mutagenesis of Amino Acids Proposed

To Function in Acid−Base Catalysis. Our pH−rate data

indicate that AANATA catalysis involves one species with a

pK_a,appof 7.0 and a second species with a pK_a,appof 9.8. The

The general base in the AANATA catalytic cycle is likely Glu-

47, Asp-142, or His-178, based on our pH−rate data, sequence

alignment, and crystal structure (Figure 4). We mutated each of

these residues to Ala to further deﬁne their respective role in

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Table 4. Steady-State Kinetic Constants for AANATA Site-Directed Mutants

Acetyl-CoA

ab

,

variant

K_m,app(μM)

k_cat,app(s⁻¹

16

)

(k_cat/K_m)_app(M⁻¹s⁻¹

)

(k_cat/K_m)_app‑mutant/(k_cat/K_m)_{app‑wild‑type}× 100 (%)

100

9.5

1.8

27

wild type

E47A

39 12

1

(4.1 1.3) × 10⁵

(3.9 0.6) × 10⁴

(7.3 0.5) × 10³

(1.1 0.1) × 10⁵

(3.4 0.3) × 10⁵

(2.5 0.4) × 10⁵

(1.5 0.1) × 10⁵

(7.3 0.3) × 10⁵

(1.7 0.3) × 10⁵

(2.1 0.2) × 10⁵

(2.6 0.3) × 10⁴

Tyramine

32

3

1.26 0.04

1.4 0.1

8.6 0.2

27.5 0.6

P48A

190 10

Y64A

81

80

5

6

D142A

R153A

H178A

C181A

S186A

83

61

430 60

106

6

120

26

7

1

5

4

17.9 0.4

19.1 0.2

5.0 0.3

8.9 0.3

4.2 0.2

37

178

41

29

S182A/S186A

H220A

43

51

160 20

6.3

ac

,

variant

K_m,app(μM)

k_cat,app(s⁻¹

)

(k_cat/K_m)_app(M⁻¹s⁻¹

)

(k_cat/K_m)_app‑mutant/(k_cat/K_m)_{app‑wild‑type}× 100 (%)

wild type

E47A

12

1

19

1

(1.6 0.2) × 10⁶

(6 2) × 10³

100

0.38

0.12

6.3

94

160 50

470 50

87 17

1.0 0.1

P48A

0.86 0.04

8.8 0.4

(1.84 0.2) × 10³

(1.0 0.2) × 10⁵

(1.5 0.1) × 10⁶

(3.4 0.3) × 10⁵

(5.9 0.8) × 10⁵

(1.3 0.1) × 10⁶

(6.9 0.6) × 10⁴

(4.3 0.3) × 10⁴

(4.9 0.6) × 10⁴

Y64A

D142A

R153A

H178A

C181A

S186A

17

1

25

98

1

2

290 20

21

25

17

3

2

14.7 0.5

22

37

1

81

73 12

5.0 0.1

8.3 0.2

4.1 0.2

4.3

2.7

3.1

S182A/S186A

H220A

190 20

82

9

a

b

Kinetic constants are reported with the standard error (n = 3). The reaction rate was measured at a ﬁxed saturating concentration of tyramine

while varying the concentration of acetyl-CoA. The reaction rate was measured at a ﬁxed saturating concentration of acetyl-CoA while varying the

c

concentration of tyramine.

Figure 5. Crystal structure of D. melanogaster AANATA with bound acetyl-CoA identifying the possible general acid residues. Stick model showing

the proximity of Tyr-64, Cys-181, Ser-182, Ser-183, and Ser-186 to acetyl-CoA. Red spheres represent water molecules.

catalysis. Mutation of Glu-47 has the greatest eﬀect on catalysis,

in that the k_cat,appvalue decreases ∼15-fold and the (k_cat/K_m)_app

decreases 10−200-fold relative to wild-type values (Table 4). In

addition, the E47A mutant exhibited the same K_{m,app‑acetyl‑CoA}

value as the wild type (within experimental error) but did show

a 10−15-fold increase in K_{m,app‑tyramine}. The D142A and H178A

mutants were catalytically competent, exhibiting k_cat,appvalues

that were 80−170% of the wild-type value. The small decreases

observed in the (k_cat/K_m)_appvalues for the D142A and H178A

mutants relative to the wild-type value resulted largely from

increases in the K_m,appvalues for both acetyl-CoA and tyramine

(Table 4).

The AANATA crystal structure also points toward Tyr-64,

Cys-181, Ser-182, or Ser-186 serving as the general acid (Figure

5) with a pK_a,appof 9.8, visible in our pH−rate studies. Each of

these residues was mutated to an Ala, and the kinetic

parameters for each mutant were determined. The steady-

state kinetic parameters for the C181A mutant are similar to

the wild-type values. In contrast with the C181A mutant, the

Y64A and S186A mutants were catalytically deﬁcient, yielding

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Figure 6. Determination of the coenzyme A dissociation constant by ﬂuorescence titration for (A) the wild-type enzyme and (B) the R153A mutant.

(k_cat/K_m)_appvalues that were 5−40% of the wild-type value, the

decrease mostly came from increases in the K_m,appvalues (Table

4). Also, we constructed and evaluated the S182A/S186A

double mutant. The kinetic parameters for S182A/S186A were

similar to those of the S186A single mutant, with the exception

of an even higher K_{m,app‑tyramine}, such that the value increased

from 12 μM for the wild type to 73 μM for the S186A mutant

and to 190 μM for the S182A/S186A double mutant (Table 4).

Site-Directed Mutagenesis of Other Amino Acids

Conserved between AANATA and Other AANAT-like

Enzymes Found in Drosophila. Three residues, Pro-48, Arg-

153, and His-220, were conserved among AANATA and the

other Drosophila AANATL enzymes (Figure S3 of the

Supporting Information) and seem necessary in maintaining

the AANATA structure.³⁷Each of these residues was mutated

to an Ala in an attempt to characterize their function (Figures

S4 and S5 of the Supporting Information). The results are

similar for both the P48A and H220A mutant enzymes,

including (k_cat/K_m)_appvalues that are 16−830-fold lower than

the wild-type value resulting from decreases in the k_cat,appvalues

(16−870-fold lower) and increases in the K_m,appvalues for the

substrates (4−40-fold higher) (Table 4). Notably, the (k_cat/

K_m)_{app‑tyramine}for the P48A mutant is 830-fold lower than that of

the wild type. The pattern of results for the R153A mutant is

diﬀerent as the (k_cat/K_m)_appvalue is decreased only 2−5-fold

relative to that of the wild type. The outcome for the R153

mutant results from a 5−7-fold increase in k_cat,appvalues that is

balanced against 10−24-fold increases in the K_m,appvalues for

the acetyl-CoA and tyramine substrates (Table 4).

Coenzyme A (CoA-SH) Binding to Wild-Type AANATA

and the R153A Mutant. The increase in k_cat,appvalues for the

R153A mutant was unexpected and may be related to a

decrease in the binding aﬃnity of the AANATA for CoA-SH,

one product of the reaction. The equilibrium constant for the

dissociation of CoA from the enzyme·CoA complex was

obtained by measuring the quenching of intrinsic protein

ﬂuorescence at diﬀerent CoA-SH concentrations for both wild-

type AANATA and the R153A mutant. From these studies, we

determined that the K_{d,wild‑type}= 400 50 μM and the K_d,R153A

= 950 110 μM (Figure 6).

DISCUSSION

■

Comparison of AANATA and AANATB. AANATA diﬀers

from AANATB by an N-terminal truncation of 35 amino acids.

Both variants are physiologically relevant in D. melanogaster, but

diﬀerences in the expression patterns for each enzyme with

respect to tissue distribution and stages of development have

been identiﬁed.²²Although the diﬀerences in the expression

patterns for the two enzymes suggest that AANATA and

AANATB do not serve the same metabolic role in D.

melanogaster, it is not known if truncation of AANATA to

AANATB has any eﬀect on the catalytic eﬃciency of the

enzyme. A comparison of the kinetic constants for AANATA

and AANATB shows that the two enzymes are catalytically

equivalent, at least for the substrates included in our study.

Thus, our data provide no clear insight regarding the

diﬀerential expression of two AANAT variants in Drosophila.

Most likely, the presence of AANATA and AANATB is

important for diﬀerences in (a) subcellular localization for the

two enzymes, (b) substrate speciﬁcities between the two

enzymes that were not revealed by our work, (c) the modes

regulation for the two enzymes, and/or (d) the post-

translational modiﬁcation of the two enzymes. Potential

diﬀerences in post-translational modiﬁcation patterns between

AANATA and AANATB could be important because the

activity of sheep serotonin N-acetyltransferase is regulated by

the phosphorylation of Thr/Ser residues located in N-terminal

or C-terminal regions of the protein.³⁸⁻⁴⁶

Salt Dependence of the Initial Rates. Arg-153 forms a

salt bridge with Asp-46, and our evaluation of the R153A

mutant indicates that the Arg-153−Asp-46 salt bridge is

important in substrate binding and catalysis. The salt

dependence of kinetic parameters, if any, could provide

additional support for this suggestion. We found an increase

in k_cat,appand K_{m,app‑acetyl‑CoA}as the concentration of NaCl added

to the reaction buﬀer increased from 0 to 500 mM [0 mM

NaCl, Table 1; 100 mM NaCl, (k_cat,app

)

= 49 1 and

acetyl‑CoA

(K_m,app

)

_acetyl‑CoA= 89 10; 500 mM NaCl, (k_cat,app

= 150 10].

)_acetyl‑CoA= 60

Substrate Speciﬁcity and the Potential Role of

AANATA in Fatty Acid Amide Biosynthesis. We have

1 and (K_m,app

)

acetyl‑CoA

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had a long-standing interest in the biosynthesis of the fatty acid

amides, a broad family of cell signaling lipids. One

straightforward route to the fatty acid amides would be the

reaction of a biogenic amine with an acyl-CoA, a possibility that

has received relatively little attention. The identiﬁcation of N-

acyltransferases that catalyze the acyl-CoA-dependent forma-

tion of N-fatty acylglycines,⁴⁷N-fatty acylserotonins,²⁷and N-

fatty acyldopamines²⁷means that this chemistry should be

examined in greater detail. Fatty acid amides are produced in D.

melanogaster,⁴⁸but the enzymes responsible for their

production in the ﬂy are largely unknown. There is a single

report of an enzyme, arylalkylamine N-acyltransferase like 2

(AANATL2), that catalyzes the formation of long-chain N-

acylserotonins and N-acyldopamines.²⁷We undertook a

detailed investigation of the substrate speciﬁcity of AANATA

to address this question.

are respectable AANATA substrates with tyramine exhibiting

the highest (k_cat/K_m)_appvalue (at saturating acetyl-CoA) and

serotonin the worst, with a (k_cat/K_m)_app,tyramine/(k_cat

/

K_m)_{app,serotonin}ratio of 6 (Table 3). The diﬀerences in the

kinetic parameters among this set of biologically relevant

arylalkylamines are reﬂected in the K_m,appvalues, whereas the

k_cat,appvalues are similar for these amino donor substrates. Our

results for the substrate speciﬁcity of AANATA with respect to

both acyl-CoA and biologically relevant arylalkylamines provide

evidence that the fatty acid amides can be produced by the

conjugation of biogenic amines to an acyl-CoA. However,

AANATA is probably not involved in the in vivo biosynthesis of

long-chain fatty acid amides because the long-chain fatty acyl-

CoA thioesters are not substrates. The major function of

AANATA in the cell is likely amine acetylation, a reaction

important in neurotransmitter inactivation, melatonin biosyn-

thesis, and cuticle sclerotization.

We ﬁrst evaluated the substrate speciﬁcity for the acyl-CoA

thioester substrates. The K_{m,app‑acyl‑CoA}values were similar for

acetyl-CoA through octanoyl-CoA, indicating that the

AANATA active site can eﬀectively bind the straight-chain

acyl chain with lengths of eight or fewer carbon atoms.

However, the k_cat,appdecreased ∼30-fold as the acyl change

length increased from acetyl-CoA to octanoyl-CoA, with acetyl-

CoA being the substrate with the highest (k_cat/K_m)_appvalue.

Lengthening the acyl chain to more than eight carbon atoms

resulted in a dramatic eﬀect on the kinetic parameters.

Decanoyl-CoA is a poor substrate relative to the shorter-

chain acyl-CoA substrates, with a K_m,appvalue of 220 μM, >10-

fold higher than that of octanoyl-CoA, and a k_cat,appvalue of

0.04 s⁻¹, 7-fold lower than that of octanoyl-CoA. Lauroyl-CoA

and longer-chain acyl-CoA thioesters are not AANATA

substrates but are inhibitors of the enzyme, with IC₅₀values

of 0.4−1.0 μM. These data show that acetyl-CoA is best

positioned for nucleophilic attack by the amino group of

tyramine within the AANATA active site and that an increase in

length alters the arrangement of the two substrates into

suboptimal positions, resulting in a decrease in k_cat,app. The

AANATA active site must have suﬃcient ﬂexibility to

accommodate the lengthening acyl chain, up to eight carbon

atoms, and still position the two substrates for chemistry with

reasonable eﬀectiveness as the (k_cat/K_m)_appfor octanoyl-CoA is

3% of the acetyl-CoA value. The limit of active site “tolerance”

is an acyl chain of 10 carbon atoms because decanoyl-CoA is a

poor substrate with a (k_cat/K_m)_appvalue that is 0.04% of the

acetyl-CoA value. Acyl-CoA thioesters with acyl chains longer

than 10 carbon atoms bind to AANATA and are not substrates;

the long-chain acyl-CoA thioesters either prevent tyramine

binding in a position that allows chemistry to occur or actually

prevent tyramine from binding. Thus, the long-chain acyl-CoA

thioesters are inhibitors of AANATA.

Our discovery of the AANATA-catalyzed acetylation of 5-

methoxytryptamine opens up an alternative route for melatonin

biosynthesis. Melatonin is produced from ^L-tryptophan in a

series of four steps: hydroxylation to 5-hydroxytryptophan,

decarboxylation to serotonin, acetylation to N-acetylserotonin,

and methylation to N-acetyl-5-methoxytryptamine (melato-

nin).^49,50Our data for the AANATA-catalyzed acetylation of

s e r o t o n i n a n d 5 - m e t h o x y t r y p t a m i n e [ ( k _{c a t}

/

K_m)_{app,5‑methoxytryptamine}/(k_cat/K_m)_{app,serotonin}∼ 2] show that both

of these amines are eﬀective AANATA substrates, similar to

that reported by Falcon et al.⁵¹It seems plausible that

melatonin biosynthesis could involve either serotonin acetyla-

tion followed by methylation or serotonin methylation followed

by acetylation. One question is whether 5-methoxytryptamine

is biosynthesized in vivo to allow direct acetylation to

melatonin. The level of 5-methoxytryptamine was shown to

increase in a time-dependent manner in hamster skin cells upon

incubation with serotonin.⁵²Further studies are required to

address this possible route to melatonin in D. melanogaster.

In addition to the biologically relevant amines, a larger group

of arylalkylamine substrates were analyzed to understand the

general structural features of the amino donor substrates that

aﬀect AANATA binding and/or catalysis. These include indole

and phenyl ring modiﬁcations, the length of the spacer group

between the amino group and the phenyl ring, modiﬁcation of

the α- and β-positions on the ethylamine spacer group, and

other non-arylalkylamines that may function as amine

substrates.

The ring-substituted analogues of tyramine included herein

are dopamine, phenethylamine, 3-(triﬂuoromethyl)-

phenethylamine, 3-methoxyphenethylamine, 4-methoxyphene-

thylamine, 3,4-methylenedioxyphenethylamine, 3,4-methoxy-

phenethylamine, and 7-methyltryptamine. All are respectable

AANATA substrates with k_cat,appvalues that are approximately

the same as that for tyramine (19 s⁻¹) or higher. The tyramine

analogue with the highest k_cat,appvalue of 71 s⁻¹is 3-

(triﬂuoromethyl)phenethylamine. However, there is consid-

erable variation in the K_m,appvalues among the tyramine series,

ranging from 12 μM for tyramine to 3200 μM for 3,4-

dimethoxyphenethylamine (Table 3). Similar trends were

observed in comparing the kinetic parameters between

tryptamine and the ring-substituted tryptamine analogue: little

variation in the k_cat,appvalues with greater diﬀerences being

found for the K_m,appvalues.

Amino donors are deﬁned herein as compounds that contain

a free amino moiety. The substrate speciﬁcity of AANATA was

evaluated using a variety of arylalkylamine amino donors, and

these data show that tyramine is the amino donor with the

highest (k_cat/K_m)_appvalue. The kinetic constants for tyramine,

with both AANATA and AANATB, were very similar (for

AANATB, K_{m,app‑tyramine}= 20 3 μM and k_{cat,app‑tyramine}= 16

1

s⁻¹compared to the data for AANATA in Table 3).

We evaluated a number of biologically important arylalkyl-

amines as AANATA substrates, including tyramine, octop-

amine, dopamine, tryptamine, norepinephrine, and serotonin.

These arylalkylamines are found in vivo and potentially could be

found as the amine partner in the fatty acid amide family. All

Tyramine and tryptamine exhibit relatively high (k_cat/K_m)_app

values, allowing us also to examine how changes in the spacer

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group between the amine moiety and the phenyl or indole ring

aﬀect AANAT catalysis. First, we determined that the cognate

amino acids, tyrosine and tryptophan, were neither substrates

nor inhibitors of AANATA, as were the results for tyrosine

methyl ester. These data demonstrate that the presence of an α-

carboxylate or α-carboxylate ester dramatically decreases

AANATA binding aﬃnity, mainly because of steric interference

in the active site. In contrast with our data about the eﬀect of α-

substitution of tyramine, we ﬁnd that modiﬁcation at the β-

position has an only small eﬀect on binding and catalysis as

octopamine, norepinephrine, and β-methylphenethylamine are

all AANATA substrates with (k_cat/K_m)_appvalues that are 20−

80% of the value for tyramine. The relatively minimal eﬀect of

β-substitution is on binding aﬃnity as all of the β-substituted

tyramines have approximately the same k_cat,appvalues. This

pattern holds true for the comparison of dopamine to

norepinephrine (β-hydroxyldopamine), which both have

approximately the same K_m,app, k_cat,app, and (k_cat/K_m)_appvalues.

Also, we interrogated the importance of the spacer length

between the amino group and the phenyl ring using another set

of tyramine analogues. Decreasing the spacer length from two

methylene groups to one methylene group yields benzylamine,

C₆H₅-CH₂-NH₂. Benzylamine is not a substrate. Increasing the

spacer length from two methylene groups to four methylenes

yields 4-phenylbutylamine, C₆H₅-(CH₂)₄-NH₂. 4-Phenylbutyl-

amine is a poor AANATA substrate, exhibiting a (k_cat/K_m)_app

that is 120-fold decreased relative to that of tyramine, with the

largest extent of the decrease reﬂected in an ∼20-fold increase

in K_m,app[12 μM for tyramine vs 270 μM for 4-phenylbutyl-

amine (Table 3)]. Our speciﬁcity studies of the amino donor

substrates again demonstrate that the AANATA active site is

ﬂexible, able to accommodate a variety of structures particularly

analogues of tyramine or tryptamine, with either a β-

substitution of the ethylamine moiety or the addition of

hydrophobic bulk to the phenyl or indole rings. AANATA is

less tolerant of substitution at the α-substitution of the

ethylamine moiety or changing the length of the methylene

spacer between the amino group and the ring. Substitution at

the α-position seems to eliminate binding to AANATA, while

alternation in the length of space hinders the optimal

positioning of the amino donor for nucleophilic attack of

acetyl-CoA.

The availability of recombinant AANATA has aﬀorded us the

opportunity to explore the speciﬁcity of this enzyme for both its

acyl-CoA and amine substrates. The structure−activity

information obtained from this work will be valuable for the

future development of inhibitors targeted against the N-

acyltransferases, particularly those involved in fatty acid amide

biosynthesis. Errors in fatty acid amide metabolism are

correlated to human disease,⁵³⁻⁵⁶and considerable eﬀort has

been devoted to the development of inhibitors for the major

enzyme responsible for fatty acid amide degradation, fatty acid

amide hydrolase (FAAH).⁵⁷

Kinetic Mechanism and Inhibitor Analysis. An inter-

secting initial velocity double-reciprocal plot indicates that a

ternary complex is formed prior to catalysis for AANATA. The

dead-end inhibitor patterns for oleoyl-CoA and tyrosol point

toward an ordered sequential mechanism, with acetyl-CoA

binding ﬁrst, followed by tyramine binding to generate the

AANATA·acetyl-CoA·tyramine ternary complex before catal-

ysis occurs. An ordered sequential mechanism with acetyl-CoA

binding ﬁrst suggests that acetyl-CoA binding drives a

conformational change to convert the amine binding pocket

from a low-aﬃnity state to a high-aﬃnity state. Oleoyl-CoA is a

known inhibitor of human and sheep serotonin N-acetyl-

transferase, and the inhibition is not a detergent eﬀect of the

C₁₈tail because the IC₅₀for oleic acid is 500-fold higher than

that for oleoyl-CoA.^58,59Tyrosol is an analogue of tyramine,

with the amine being replaced with a hydroxyl group. A rate

was not observed for tyrosol acetylation at 100 mM, which

shows that AANATA will not catalyze the O-acylation of at

least this substrate. Tryptophol, a similar analogue of

tryptamine, is an inhibitor of sheep serotonin N-acetyltransfer-

ase and was useful in studies to deﬁne the kinetic mechanism of

this enzyme.⁶⁰An ordered sequential mechanism for D.

melanogaster AANATA is consistent with isothermal calorim-

etry data demonstrating little binding of dopamine to AANATA

in the absence of acetyl-CoA,³⁷as well as with the kinetic

mechanism proposed for sheep serotonin N-acetyltransferase.⁶⁰

Site-Directed Mutagenesis To Match Catalytic Amino

Acids to the Measured pK_a,appValues. We employed

protein sequence alignments, pH−rate studies, site-directed

mutagenesis of targeted amino acids, and information from the

AANATA crystal structure³⁷to propose a chemical mechanism

for the AANATA-catalyzed formation of N-acylarylalkylamides.

Primary sequence alignment of AANATA with other D.

melanogaster AANATL enzymes identiﬁed a number of

conserved residues that might function in catalysis. A primary

sequence alignment of D. melanogaster AANATA with sheep

serotonin N-acetyltransferase produced a low sequence align-

ment score (<12%), and the catalytic core of the mammalian

ortholog⁶¹⁻⁶⁶is not conserved in the ﬂy enzyme. Therefore,

AANATA could catalyze the formation of the same N-

acetylarylalkylamide products as the mammalian ortholog

with either a completely diﬀerent chemical mechanism or, at

minimum, diﬀerent catalytic amino acid residues.

Our pH−rate studies implicate two chemical species with

pK_a,appvalues of 7.0 and 9.8 in AANATA catalysis (Figure 3).

The pK_a,appvalue of 7.0 was observed in the k_cat,appand (k_cat/

K_m)_appproﬁles and, most likely, reﬂects deprotonation by Glu-

47. Evidence consistent with the proposal that Glu-47 serves as

a general base in the AANAT catalytic cycle includes the

conservation of Glu-47 among the D. melanogaster family of

AANATL enzymes, the catalytic deﬁciency of the E47A mutant,

and the disappearance of the pK_a,appof 7.0 in the pH−rate

proﬁle of the E47A mutant. Cheng et al.³⁶reported that the

log(k_cat/K_m)_appversus pH proﬁle for the E47A mutant is linear,

with a slope of 0.3, probably resulting from hydroxide

chemically rescuing Glu-47 as the base in AANATA catalysis.

Furthermore, a Glu residue has been proposed as the general

base in other GNAT enzymes such as members of the Gcn5/

PCAF family of histone N-acetyltransferases^67,68and human

spermidine/spermine N¹-acetyltransferase.⁶⁹⁻⁷¹

A pK_a,appof 9.8 was observed in the pH−rate proﬁles for only

k_cat,app, indicating that this chemical species contributes to

catalysis only after the ﬁrst irreversible step (Figure 3A,B). We

must point out that Ellman’s reagent is unstable and the acyl-

CoA substrates are subject to rapid base-catalyzed hydrolysis at

pH >9.5. Because we measure AANAT activity by assaying CoA

release using Ellman’s reagent, our pH−rate studies are

conﬁned to pH ≤9.5. Thus, the lack of a pK_a,appof ≥9.8 in

our (k_cat/K_m)_appproﬁles may reﬂect our inability to conduct

measurements at pH >9.5, and the presence of a pK_a,appof 9.8

for in the k_cat,appproﬁles is based on a ﬁt of our data, all

generated at pH ≤9.5, to eq 9. Cheng et al.³⁷extended their

studies to pH 10 and found a clearly deﬁned pK_a,appof 9.0 0.2

K

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Figure 7. Crystal structure of D. melanogaster AANATA with bound acetyl-CoA showing a possible “proton wire” involving the general base, Glu-47.

a

Scheme 1. Proposed Chemical Mechanism for D. melanogaster AANATA

a

R has diﬀerent acyl-chain lengths for acyl-CoA. The dashed arrows represent ambiguities in the position of the irreversible step caused by

diﬀerences between the pH−rate proﬁles reported herein and those reported by Cheng et al.³⁷

in their pH−rate proﬁles for (k_cat/K_m)_app. Experimental

diﬀerences between our work and that of Cheng et al.³⁷render

direct comparisons of the respective data sets diﬃcult, but it

seems likely that a chemical species with a pK_aof 9−10 does

participate in AANATA catalysis.

With this in mind, we sought to identify the chemical species

that would serve as a general acid in AANATA; possibilities

include Tyr-64, Cys-181, Ser-182, Tyr-185, and Ser-186 (Figure

5). Cys-181 and Tyr-185 were eliminated because the

corresponding alanine substitution mutants (C181A and

Y185A) showed little to no eﬀect on the k_cat,apprelative to

the wild-type value (Table 4 and ref 36). Tyr-64 is located

within a nonpolar binding pocket for the amino donor

substrate,³⁷and comparison of the Y64A mutant to the wild

type reveals increases in the K_m,appvalues for both substrates (2-

fold for acetyl-CoA and 6-fold for tyramine) and an only ∼2-

fold decrease in the k_cat,appvalues (Table 4). Most likely, Tyr-64

is involved in substrate binding and is not the general acid in

AANATA catalysis. Ser-182 and Ser-186 remain as potential

general acids. The mutation of both to Ala decreases k_cat,app

relative to that of the wild type, 12-fold for the S182A mutant

and 3−5-fold for the S186A mutant (Table 4 and ref 36). The

pH−rate proﬁles for the S182A and S186A mutants are pH-

independent³⁷at pH ≥7.0, meaning that the pK_a,appof 9.0 is

not observed for these two mutant enzymes. On the basis of

these data and the proximity of both Ser-182 and Ser-186 to the

L

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acetyl-CoA thiol group, 4.0 and 4.1 Å, respectively (Figure 7), it

is conceivable that either Ser-182 or Ser-186 is the general acid

in AANATA catalysis. In fact, Cheng et al.³⁷argue that Ser-186

is the general acid, functioning to protonate the departing

thiolate anion of CoA to complete the catalytic cycle. An

argument could be made that Ser-182 and Ser-186 serve

redundant roles in catalysis where one of these serines is the

general acid, whereas the other serine serves to depress the pK_a

of the catalytic serine. In the absence of the catalytic serine, the

other serine could chemically “rescue” the function of the other

serine.

We do not favor either Ser-182 or Ser-186 being the general

acid in AANATA catalysis. This would require a signiﬁcant

depression of 3−5 pH units in the pK_afor Ser-182 or Ser-186,

and we would expect the S182A/S186A double mutant to be

dramatically impaired as a catalyst relative to either the S182A

or S186A single mutant. Instead, we ﬁnd that the S182A/

S186A double mutant exhibits a k_cat,appsimilar to that of the

S186A mutant; the largest diﬀerence for S182A/S186A is an

increased K_m,appfor tyramine. A tyrosine serves as the general

acid for other GNAT enzymes, but the mutation studies

eliminate the two active tyrosine residues, Tyr-64 and Tyr-185,

as possibilities. There are no other obvious candidate amino

acids to serve as a general acid within the acetyl-CoA binding

pocket.³⁷

We propose that the pK_a,appof 9.8 in our pH−rate proﬁles

(and the pK_a,appof 9.0 from ref 37) represents the pK_aof the

thiol group of CoA, which has been reported to range from 9.6

to 10.4.^72,73Formation of the protonated CoA-SH product

results from the collapse of the tetrahedral intermediate

(Scheme 1), as proposed for other N-acyltransferases.⁷⁴⁻⁷⁶

The deprotonation of CoA-SH to CoA-S⁻, in the AANATA

active site, inhibits product release, accounting for the decrease

in k_cat,appat pH >9. This was likely not observed in the (k_cat/

K_m)_appproﬁles because of either the diﬃculty in measuring the

kinetic constants at high pH (instability of DTNB and/or the

rapid hydrolysis of acetyl-CoA) or the fact that the protonation

of CoA-S⁻occurs after the ﬁrst irreversible step in catalysis.

Our suggestion obviates the need for an active site residue that

serves as the general acid and is consistent with the data

available for AANATA.

Site-Directed Mutagenesis of Amino Acids That Are

Important in Substrate Binding and AANAT Structure.

As detailed previously, we mutated a number of amino acids in

AANATA in an attempt to match an active site amino acid to

the pK_a,appvalues identiﬁed in the pH−rate proﬁles. The set of

AANATA mutants that we produced also provide information

regarding the amino acids involved in substrate binding and

point toward structural changes in the protein. In silico docking

of dopamine into the AANATA structure with acetyl-CoA

bound suggests that the amino donor substrate binds into a

hydrophobic binding pocket containing Phe-43, Leu-61, Tyr-

64, Ile-116, and Ile-145.³⁷Our results are consistent with the

docking results as the K_{m,app‑tyramine}values for the E47A, P48A,

and Y64A mutants are >6-fold higher than the wild-type value

(Table 4). The K_{m,app‑tyramine}values for H220A and S186A single

mutants also are ≥6-fold higher than the wild-type value,

indicating that these amino acids contribute to the binding of

the amino donor substrate. Of the mutants with a 6-fold

increase in the K_{m,app‑tyramine}value relative to the wild-type value,

only the E47A and S186A mutants showed no diﬀerence in

S182A/S186A double mutant (190 μM) is signiﬁcantly higher

than the wild-type value (12 μM), suggesting that Ser-182 and

Ser-186 function synergistically in maintaining the proper active

site architecture for optimal binding to the amino donor

substrate. The binding pocket for acetyl-CoA is more extensive

than the pocket for the amino donor substrate and includes the

amino acids involved in catalysis.

There are amino acids uniquely involved in acetyl-CoA

binding, for which there is a change of only the value of

K_{m,app‑acetyl‑CoA}upon mutation. This was observed for the

D142A and H178A mutants: K_{m,app‑tyramine}values that are

approximately the same as that of the wild type and

K_{m,app‑acetyl‑CoA}values that are 2−3-fold higher than that of the

wild type. Both K_{m,app‑tyramine}and K_{m,app‑acetyl‑CoA}values for the

P48A, Y64A, R153A, and H220A mutants are all diﬀerent from

and greater than the wild-type values (Table 4). These amino

acids either are involved in domains within AANATA that

overlap between the acetyl-CoA and amino donor binding

pockets or are key to maintaining the AANATA structure in a

conformation that is optimal for substrate binding. Overlap

between the binding sites for the two substrates is expected

because the binding pocket for acetyl-CoA is more extensive

than the pocket for the amino donor substrate³⁶and should

include the amino acids that are required to position the amino

group of the amino donor for nucleophilic attack at the

carbonyl of the acetyl-CoA thioester moiety. In addition, the

synergistic eﬀect of acetyl-CoA binding to convert the amine

binding pocket from a low-aﬃnity state to a high-aﬃnity state

means that speciﬁc amino acids important for acetyl-CoA

binding can also have a dramatic eﬀect on amine binding.

In comparing the kinetic constants of our mutants to those of

wild-type AANATA (Table 4), we ﬁnd that three of our

mutants stand out: E47A, P48A, and R153A. We argue that

Glu-47 is the catalytic base and ﬁnd, as anticipated, that E47A is

catalytically deﬁcient with relatively low k_cat,appvalues. The

AANATA structure containing bound acetyl-CoA³⁷reveals a

set of ordered water molecules between the carboxylate of Glu-

47 and the carbonyl of the acetyl-CoA thioester. The distance

from the carboxyl of Glu-47 to the carbonyl of the acetyl-CoA

thioester is 6.8 Å. We suggest that Glu-47 functions as a general

base to allow the water-assisted deprotonation of the positively

charged amino group of the donor substrate (a “proton wire”)

and to properly position the neutral amine for attack at the

carbonyl of the acetyl-CoA thioester (Scheme 1). Elimination

of the anionic carboxylate of Glu-47 by mutation to Ala would

alter the positions of the ordered water molecules (perhaps,

even resulting in their loss) and would decrease the aﬃnity for

only the amino donor substrate, as found for the E47A mutant.

Pro-48 is found at the beginning of a ﬂexible loop and is

positioned within the predicted binding pocket for the amine

donor substrate (Figure S6 of the Supporting Information). We

superimposed the AANATA crystal structure [Protein Data

Bank (PDB) entry 3TE4]³⁷with that of sheep serotonin N-

acetyltransferase (PDB entry 1CJW)⁶¹using Protein Binding

Site Tools (ProBiS Tools) and found that Pro-48 from D.

melanogaster AANATA is equivalent to Pro-64 from sheep

serotonin N-acetyltransferase. In sheep serotonin N-acetyl-

transferase, Pro-64 is also positioned on a ﬂexible loop that

undergoes a major structural change to alter the amino donor

substrate binding pocket from a low- to high-aﬃnity state.^77,78

In the low-aﬃnity state, Pro-64 occupies the acetyl-CoA

binding pocket, whereas in the high-aﬃnity state, Pro-64 moves

∼8 Å to form a base-staking interaction with the tryptamine

K_{m,app‑acetyl‑CoA}, indicating that Glu-47 and Ser-186 contribute

little to acetyl-CoA binding. The K_{m,app‑tyramine}value for the

M

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moiety of the tryptamine−acetyl-CoA bisubstrate inhibitor

cocrystallized with the sheep enzyme. Our data for the P48A

mutant, low k_cat,appand high K_{m,app‑tyramine}and K_{m,app‑acetyl‑CoA}

values, suggest that Pro-48 in AANATA functions like Pro-64 in

sheep serotonin N-acetyltransferase and further hints that

protein dynamics could regulate catalysis for AANATA. His-

220 is in van der Waals contact with Pro-48 in AANATA

(Figure 8). Replacement of His-220 with Ala will alter this

CoA bound,³⁷Arg-153 participates in a water-mediated

hydrogen bond with the carbonyl amide of the pantothenate

moiety (atom O9P) of acetyl-CoA and in a salt bridge to Asp-

46 (Figure 9). The increase in the K_m,appvalues for both

substrates suggests that Arg-153 is critical not only in the

acetyl-CoA binding pocket but also in forming the binding

pocket for the amine substrate. The increase in the k_cat,appvalue

suggests that Arg-153 contributes to a rate-determining step in

catalysis, related to the salt bridge between Arg-153 and Asp-46.

We suspect that the Arg-153−Asp-46 salt bridge is critical to an

AANATA conformation that decreases the rate of CoA-SH

release, such that product release is, at least, partially rate-

limiting. Partially rate-determining CoA-SH release has been

identiﬁed in many other N-acetyltransferases.^20,66,79,80Elimi-

nation of the Arg-153−Asp-46 salt bridge in the R153A

increases the rate of CoA-SH release, reﬂected in the increase in

k

_cat,app, but decreases the aﬃnity of AANATA for both

substrates, reﬂected in the increases in the K_m,appvalues.

Another approach to decrease the inﬂuence of the Arg-153−

Asp-46 salt bridge on the rate of CoA-SH release would be to

increase the salt concentration in the reaction buﬀer. Our

prediction is that a greater salt concentration would mimic the

pattern of results we obtained for the R153A mutant, which is

exactly what we found. The values of k_cat,appand K_{m,app‑acetyl‑CoA}

increased as the concentration of added NaCl increased from 0

to 500 mM. Similar data were obtained for sheep serotonin N-

acetyltransferase,²⁵which was argued to increase the rate of

CoA-SH release by shielding charge−charge interactions of the

enzyme with the anionic phosphates of acetyl-CoA. Also, we

determined the K_dfor binding of CoA-SH to both wild-type

AANATA and the R153A mutant by measuring the quenching

of intrinsic protein ﬂuorescence as a function of CoA-SH

concentration [K_{d,wild‑type}= 400 50 μM, and K_d,R153A= 950

110 μM (Figure 6)]. The 2.4-fold decrease in aﬃnity for the

binding of CoA-SH to the R153A mutant is in reasonable

Figure 8. Crystal structure of D. melanogaster AANATA depicting the

structural importance of His-220. CPK schematic of His-220 found in

AANATA along with secondary structure highlighting van der Waals

interactions between His-220 of Tyr-185 and His-220 of Pro-48.

interaction and, as seen in the data for the P48A mutant, would

lead to the observed increases in the K_{m,app‑tyramine}and

K_{m,app‑acetyl‑CoA}values for the H220A mutant.

The R153A mutant is intriguing, with values higher than

wild-type values for k_cat,app, K_{m,app‑tyramine}, and K_{m,app‑acetyl‑CoA}. The

∼6-fold increase in k_cat,appis balanced against 10−20-fold

increases in the K_m,appvalues, such that the (k_cat/K_m)_appvalue

for the R153A mutant is lower than that of the wild type by

40−80%. As revealed from the AANATA structure with acetyl-

agreement with the (k_cat,app

)

_wild‑type/(k_cat,app

)

ratio of 5−6

R153A

(Table 4). The salt-dependent increase in both k_cat,appand

K_{m,app‑acetyl‑CoA}and the increase in K_dfor CoA-SH for the

Figure 9. Crystal structure of D. melanogaster AANATA with bound acetyl-CoA depicting the Asp-46−Arg-153 salt bridge. Stick model showing the

proximity of Asp-46 and Arg-153 to acetyl-CoA. Red spheres represent water molecules.

N

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R153A mutant provide evidence that the salt bridge between

Arg-153 and Asp-46 is important for decreasing the rate of

release of CoA-SH from wild-type AANATA. Similar to our

data for the P48A mutant, our data for the R153A mutant point

toward protein dynamics regulating catalysis for AANATA.

Chemical Mechanism for AANATA catalysis. A chemical

mechanism for the catalytic cycle for AANATA, consistent with

our new data and previously published data,^21,37is illustrated in

Scheme 1. The mechanism features (a) chemistry occurring

after the formation of the AANATA·acetyl-CoA·tyramine

complex, (b) Glu-47 serving as a general base to facilitate

water-assisted deprotonation of tyramine, (c) nucleophilic

attack of deprotonated tyramine at the carbonyl of the acetyl-

CoA thioester to form a zwitterionic tetrahedral intermediate,

(d) collapse of the tetrahedral intermediate with the

concomitant protonation of CoA, and (e) ordered product

release with CoA-SH departing last to regenerate AANATA for

the next round of catalysis. Our only evidence of the ordered

product release is the data showing that CoA-SH binds to

AANATA in the absence of N-acetyltyramine (Figure 7) and is

consistent with the ordered binding of substrates, acetyl-CoA

binding ﬁrst. The release of CoA-SH is partially rate-

determining because of the AANATA conformation that has

a relatively high aﬃnity for CoA-SH. The Arg-153−Asp-46 salt

bridge is critical to maintaining AANATA in this conformation,

and the aﬃnity for CoA-SH increases as the CoA-S⁻thiolate

forms at higher pH.

One issue remaining with this mechanism is the roles played

by Ser-182 and Ser-186 in catalysis, given that Ser-186 was

proposed to serve as the general acid in AANATA catalysis.³⁷A

preponderance of the data for the S182A, S186A, and S182A/

S186A mutants seem to rule out a direct catalytic role for both

Ser-182 and Ser-186, and the transfer of a proton from a serine

hydroxyl to a thiol is thermodynamically unfavorable. Instead,

we suggest that Ser-182 and Ser-186 are involved in an

elaborate network of hydrogen bonds that function in both

substrate binding and catalysis (Figure 4). Mutation of Ser-182

and/or Ser-186 to alanine alters the hydrogen bonding

network, changing the active site architecture. This alteration

of the active site is reﬂected in the changes observed in the

k_cat,appand K_m,appvalues for the S182A, S186A, and S182A/

S186A mutants.

site amino acids, Asp-46, Pro-48, Arg-153, and His-220, seem to

be critical to the regulatory dynamics of the enzyme.

ASSOCIATED CONTENT

* Supporting Information

■

S

Mutant PCR primers (Table S1), LC/QTOF-MS data for

product characterization (Table S2), and a list of arginines

found in other GNAT enzymes that are conserved with Arg-

153 (Table S3), and ﬁgures showing cloning and puriﬁcation

data, mutant SDS−PAGE gels, a D. melanogaster AANATL

primary sequence alignment, and the AANATA active site

depicting the location of Pro-48 with respect to bound acetyl-

CoA. This material is available free of charge via the Internet at

http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Department of Chemistry, 4202 E. Fowler Ave., CHE 205,

Tampa, FL 33620-5250. E-mail: merkler@usf.edu. Phone:

(813) 974-3579. Fax: (813) 974-3203.

■

Present Address

^⊥A.-M.C.: University of Florida College of Medicine, M-108

Health Science Center, P.O. Box 100216, Gainesville, FL

32610-0216.

Funding

This work has been supported, in part, by grants from the

Florida Center for Excellence for Biomolecular and Targeted

Therapeutics (FCoE-BITT Grant GALS020), the University of

South Florida (a Research Scholarship grant from the College

of Arts and Sciences), the Shirley W. and William L. Griﬃn

Foundation, the National Institute of Drug Abuse of the

National Institutes of Health (Grant R03-DA034323 to

D.J.M.), and a Dissertation Completion Fellowship from the

Oﬃce of Graduate Studies at the University of South Florida to

D.R.D.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Support for this work has been provided by the Mass

Spectrometry and Peptide Facility, Department of Chemistry,

University of South Florida. We thank Elisabeth Nevins

Caswell for a thorough reading and revision of the manuscript.

CONCLUSIONS

■

ABBREVIATIONS

We determined that D. melanogaster AANATA will catalyze the

formation of N-acylarylalkylamides from a broad array of

corresponding acyl-CoA and arylalkylamine substrates. These

data validate the potential role of N-acyltransferases in fatty acid

amide biosynthesis and provide structure−activity data for the

future development of inhibitors to this class of enzymes. A

chemical mechanism for AANATA, consistent with all the data

available for this enzyme,^21,37is illustrated in Scheme 1.

Substrate binding is sequential ordered with acetyl-CoA

binding ﬁrst, meaning that catalysis occurs only after the

formation of an AANAT·acetyl-CoA·arylalkylamine ternary

complex. We identiﬁed Glu-47 as the general base involved

in the water-assisted deprotonation of the positively charged

amino group of the donor substrate via an active site “proton

wire”. The deprotonation of a species with a pK_a,appof 9.8 was

attributed to the formation of the tightly bound CoA thiolate.

Finally, our mutagenesis data suggest that protein dynamics

regulate substrate binding and catalysis in AANATA. The active

■

GNAT, GCN5-related N-acetyltransferase; AANATL, arylalkyl-

amine N-acetyltransferase like enzyme; LC/QTOF-MS, liquid

chromatography/quadrupole time-of-ﬂight mass spectroscopy;

MES, 2-(N-morpholino)ethanesulfonic acid; AMeP, 2-amino-2-

methyl-1-propanol hydrochloride; Tris, tris(hydroxymethyl)-

aminomethane; IC₅₀, half-maximal inhibitory constant.

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Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases

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