Structural insights and functional implications of choline acetyltransferase

Journal of

Structural

Journal of Structural Biology 148 (2004) 226–235

Biology

www.elsevier.com/locate/yjsbi

Structural insights and functional implicationsof choline acetyltransferase

Lakshmanan Govindasamy,a Brenda Pedersen,b Wei Lian,b,c Thomas Kukar,b

Yunrong Gu,b Shouguang Jin,c Mavis Agbandje-McKenna,a Donghai Wu,b,d,*

and Robert McKennaa,*

a Department of Biochemistry and Molecular Biology, McKnight Brain Institute and University of Florida, Gainesville, FL 32610, USAb Department of Medicinal Chemistry, McKnight Brain Institute and University of Florida, Gainesville, FL 32610, USA

c Department of Microbiology and Molecular Genetics, McKnight Brain Institute and University of Florida, Gainesville, FL 32610, USAd Shanghai Institute of Nutritional Sciences, Shanghai, China

Received 8 March 2004, and in revised form 1 June 2004

Available online 3 August 2004

Abstract

The biosynthetic enzyme for the neurotransmitter acetylcholine, choline acetyltransferase (ChAT) (E.C. 2.3.1.6), is essential for

the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. ChAT catalyzes the

transfer of an acetyl group from acetyl-coenzyme A to choline to form the neurotransmitter acetylcholine. Since its discovery more

than 60 years ago much research has been devoted to the kinetic studies of this enzyme. For the first time we report the crystal

structure of rat ChAT (rChAT) to 1.55�A resolution. The structure of rChAT is a monomer and consists of two domains with an

interfacial active site tunnel. This structure, with the modeled substrate binding, provides critical insights into the molecular basis for

the production of acetylcholine and may further our understanding of disease causing mutations.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Choline acetyltransferase; Acetylcholine; Neurotransmitter; X-ray structure; Congenital myasthenic syndrome with episodic apnea

1. Introduction

Choline acetyltransferase (ChAT,1 E.C. 2.3.1.6) is the

enzyme responsible for catalyzing the biosynthesis of the

neurotransmitter acetylcholine (ACh) from its precur-

sors, acetyl-coenzyme A (acetyl-CoA) and choline, as

shown below.

* Corresponding authors. Fax: 1-352-392-3422 (R. McKenna).

E-mail addresses: [email protected] (D. Wu), [email protected]

(R. McKenna).1 Abbreviations used: ChAT, choline acetyltransferase; ACh, ace-

tylcholine; CoA, coenzyme A; CMS–EA, congenital myasthenic

syndrome with episodic apnea; CAT, LL-carnitine acetyltransferase;

E2p, dihydrolipoyl transacetylase; RMS, root mean square.

1047-8477/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.jsb.2004.06.005

ACh was the first neurotransmitter to be reported

(Loewi, 1921) and has since been shown to play a piv-

otal role in fundamental brain processes such as learn-

ing, memory, and sleep (Aigner and Mishkin, 1986;Bartus et al., 1982; Karczmar, 1993). ACh functions in

the cholinergic neurons of the peripheral and central

nervous systems. In the peripheral nervous system ACh

stimulates muscle contraction and in the central nervous

system ACh facilitates learning and short-term memory

formation. The cholinergic neurons have been impli-

cated in the pathophysiology of several diseases such as

amyotrophic lateral sclerosis, Huntington’s disease,schizophrenia, sudden infant death syndrome, and

Alzheimer’s disease (Oda, 1999).

Although ChAT has been known as the enzyme re-

sponsible for the synthesis of ACh for more than 60

years, its mechanism of action is still unclear (Oda,

1999). Several inactivation and modification studies of

ChAT have suggested several histidine and arginine

mail to: [email protected]

L. Govindasamy et al. / Journal of Structural Biology 148 (2004) 226–235 227

residues that are critical for the enzyme to function(Carbini et al., 1990; Hersh et al., 1979; Malthe-So-

renssen, 1976; Mautner et al., 1981; Roskoski, 1974a,b;

Roskoski et al., 1975). Site-directed mutagenesis studies

in Drosophila ChAT (dChAT) have lead to the proposal

that a highly conserved histidine residue, histidine 426,

(histidine 334 in rChAT) is essential for activity and may

serve as an acid/base catalyst (Carbini and Hersh, 1993).

Additional mutagenesis studies in rChAT have alsosuggested that another highly conserved residue, argi-

nine 452, interacts with acetyl-CoA (Wu and Hersh,

1995). Interestingly, an equivalent mutation in human

ChAT (hChAT), arginine 560 to histidine, has also been

shown to significantly reduced the affinity for both

substrates (Ohno et al., 2001).

Recently, direct sequencing of hChAT from five pa-

tients, exhibiting congenital myasthenic syndrome withepisodic apnea (CMS–EA), revealed 10 recessive muta-

tions. Kinetic analysis of nine of these mutants (the

tenth was a frameshift null mutation), demonstrated

that one mutant (Glu441Lys; residue 332 in rChAT)

lacked catalytic activity while the others (Leu210Pro,

Pro211Ala, Ile305Thr, Arg420Cys, Arg482Gly, Ser498Leu,

Val506Leu, and Arg560His; residues 102, 103, 197, 312,

374, 389, 398, and 453 in rChAT, respectively) hadsignificantly impaired catalytic efficiencies (Ohno et al.,

2001). Understanding the basis of these mutations,

however, has been hindered since no three-dimensional

structure was available.

Here, we report the X-ray crystal structure of rChAT

to 1.55�A resolution, determined by molecular replace-

ment using the recently determined human peroxisomal

LL-carnitine acetyltransferase (hpCAT) structure (Wuet al., 2003). The structure, together with the modeled

substrate (choline and acetyl-CoA) complexes has given

rational to understanding the mechanisms of catalysis of

the enzyme.

2. Materials and methods

2.1. Crystallization and data collection

Production, purification, and crystallization of

rChAT were performed as described previously (Lian

et al., 2004). Briefly, crystals of rChAT were obtained

by vapor diffusion crystallization procedures mixing

3 lL of 10mg/mL rChAT and 6 lL reservoir solution

[50mM Mes buffer (pH 6.0), 100mM NaCl, and 8–10% PEG 8000] at 277K. X-ray diffraction data were

collected at 100K on the F1 beam line at the Cornell

High Energy Synchrotron Source using a quantum

four CCD detector system (Lian et al., 2004). The

images were indexed, integrated, and scaled with the

DENZO and SCALEPACK programs (Otwinowski

and Minor, 1997).

2.2. Structure determination and refinement

The structure of rChAT was solved by molecular re-

placement procedures (Rossmann, 1990), with the coor-

dinates of hpCAT (with the side-chains of all non-glycine

residues truncated to alanines) as a search model (Wu

et al., 2003) using the CNS program package (Brunger

et al., 1998). Refinement followed a cyclic protocol with

simulated annealing, individual B-factor, and energyminimization procedures within CNS (Brunger et al.,

1998), interspersed with rounds of manual model build-

ing of side-chains and water placement using the graphics

program O (Jones et al., 1991). A subset of 5% of ran-

domly selected reflections were flagged and used for Rfree

calculations (Brunger, 1992). The final cycles of refine-

ment were performed with the REFMAC5 (Murshudov

et al., 1999) program. The quality of the final refinedstructure was validated with the PROCHECK (Laskow-

ski et al., 1993) and CNS (Brunger et al., 1998) programs.

2.3. Least-squares superimpositions

The least-squares superimposition of rChAT to the

hpCAT, mCAT, and dihydrolipoyl transacetylase (E2p)

structures (Jogl and Tong, 2003; Mattevi et al., 1993;Wu et al., 2003) were performed using the program O

(Jones et al., 1991).

2.4. Molecular modeling

The structures of choline and CoA were obtained

from Uppsala Factory HIC–UP database (Kleywegt

and Jones, 1998) and energy minimized prior to dockinginto the rChAT structure. Automated docking of both

substrates were performed with AUTODOCK 3.0

(Morris et al., 1998). The docking grid was centered at

the initial site of LL-carnitine and CoA, implied from a

comparison with the mCAT and hpCAT complex

structures (Govindasamy et al., 2004; Jogl and Tong,

2003), with choline replacing LL-carnitine.

2.5. Sequence alignments

The multiple sequence alignment of rat, human,

mouse,pig,Caenorhabditis elegans, andDrosophilaChATs

(GenBankTM Accession Nos. A48319, A60202, B43777,

A39961, T37293, and A36526, respectively) was obtained

using the program ClustalW (Thompson et al., 1994).

3. Results

3.1. Data collection and reduction

The rChAT crystal was shown to be orthorhombic

P212121 with unit-cell parameters a ¼ 139:0, b ¼ 77:7,

Table 1

Refinement statistics for rChAT

Resolution (�A) 30–1.55

Rsyma 0.054 (0.337)

Rfactorb 0.171(0.254)

Rfree 0.227(0.325)

Number of reflections 72 446(4106)

Average I=Ir 10.5(3.2)

Completeness (%) 77.5 (65.6)

Number of protein atoms 4714

Number of waters 408

RMS deviation, bond lengths (�A) 0.01

RMS deviation, bond angles (�) 1.81

Average B factor, main/side chains (�A2) 20.6/25.9

Average B factor for waters (�A2) 29.3

% Residues in the most allowed/additional

allowed/generously allowed/disallowed

regions of Ramachandran plot (%)

85.9/13.5/0.2/0.4

Values given in parentheses correspond to those in the outer most

shell 1.59–1.55�A.aRsym is defined as

PjI � hIij=

PI, where I is the intensity of an

individual reflection and hIi is the average intensity for this reflection.bRfactor ¼

PkFoj � jFck=

PjFoj, where Fo and Fc are the observed

and calculated structure factors, respectively.

228 L. Govindasamy et al. / Journal of Structural Biology 148 (2004) 226–235

and c ¼ 59:7�A The merged and scaled diffraction dataset consisted of 42 9316 total observations (72 446 in-

dependent reflections) with an overall Rsym of 5.4% with

77.5% completeness for data between 30.0 and 1.55�Aresolution (Table 1) (Lian et al., 2004). Using the unit-

cell parameters and the molecular weight of one rChAT

the calculated VM (Matthews, 1968) of the crystal was

2.3�A3/Da, indicating that a single rChAT molecule was

present in the crystallographic asymmetric unit.

3.2. Structure determination and refinement

A pairwise sequence alignment between rChAT and

hpCAT showed there was �40% amino acid identity

indicating the overall structural fold of the two enzymes

would be similar. A cross-rotation function (DeLano

and Brunger, 1995; Tong and Rossmann, 1997) searchwas calculated using the poly-alanine hpCAT coordi-

nates with data from 15.0 to 4.0�A resolution (Wu et al.,

2003). This resulted in a single (7.5rÞ solution. Using

this orientation matrix a translation function search

(Brunger, 1990) gave a unique solution (with Eulerian

rotation angles of h1 ¼ 97:27, h2 ¼ 0:37, and

h3 ¼ 262:47� and fractional translations of Tx ¼ 0:28,Ty ¼ 0:11, and Tz ¼ 0:02) with a correlation coefficientof 0.60. Rigid body refinement optimized the orientation

and translation position of the poly-alanine search

model and gave an initial Rwork of 44% (Rfree ¼ 45:3%)

for data from 30.0 to 2.0�A resolution. The initial cal-

culated electron density maps were of excellent quality,

the main-chain electron density was continuous, and

side-chain electron density was in agreement with the

sequence of rChAT. The side-chains of rChAT wereassigned and built using the graphics program O (Jones

et al., 1991). After one further cycle of refinement theRwork subsequently decreased to 33.0% (Rfree ¼ 34:6%).

Additional rounds of refinement using all data to

1.55�A resolution, reduced the Rwork further to 25.3%

(Rfree ¼ 27:2%). At this stage of refinement water mole-

cules, with peaks greater than 2r and within acceptable

hydrogen bonding distances, were included into the

model. This resulted in 408 water molecules placed in the

structure with a Rwork of 24.2% (Rfree ¼ 25:6%). The finalrounds of refinement, with no additional changes to the

model, were performed using the REFMAC5 program

(Murshudov et al., 1999). After iterative cycles of re-

finement the final Rfactor was 17.1% (Rfree ¼ 22:7%) (Table

1). Residues 150–154, 185–193, and 360–367 were disor-

dered. Over 85.0% of the residues in the structure are in

the most favorable region of the Ramachandran plot as

calculated by PROCHECK (Laskowski et al., 1993). Thecoordinates of rChAT have been deposited in the protein

database and assigned the Accession No. 1T1U.

3.3. rChAT structure

The structure consists of �40% helix (20 helices: H1–

H20) and �15% strand (16 strands: S1–S16) arranged

into two domains, I (residues 89–400) and II (residues20–88 and 401–616). These domains are interconnected

and create a tunnel, �16�A in diameter that passes

through the center of the enzyme, which forms the active

site (Fig. 1A).

Domain I is composed of a six-stranded mixed b-sheet (S1, S8, S7, S6, S4, and S5) that is interconnected

on both flanks by eight a-helices. Domain II also con-

sists of six-mixed b-sheets (S10, S16, S15, S13, S11, andS12) that are connected with 11 a-helices (seven from the

C-terminal and four from the N-terminal). Helices H1

and H2 generate an anti-parallel bundle that elongates

to an extended structure composed of helices H3 and

H4, which crosses over to domain I to form the front

entrance (Fig. 1A choline binding site) of the tunnel. In

domain I residues 322–335 generate anti-parallel b-sheets (S7 and S8) with the catalytic His334 located at theend of strand S8 positioned at the center of the active

site tunnel. This turn (residues 333–358) extends to form

helix H12 that lines one side of the active site tunnel.

The long helix H13 is arranged in front of the b-sheet ofdomain I and connects to b-strand S10 that links do-

main I to domain II. Strand S10 leads into helix H14

that is parallel with the b-sheet of domain II. The b-sheet of domain II facilitates the flipping out of strandS14 (between S13 and S15), which forms an anti-parallel

interaction with the strand S1 of domain I. Five helices,

H15–H20, are between strands S11 and S16, generate a

supporting scaffold that holds domain II b-sheet open

face towards the active site of the enzyme. The b-strandS16 connects with the C-terminal helix H20 via a loop

that terminates domain II. The two domains of rChAT

Fig. 1. Structure of the rat choline acetyltransferase (rCHAT). (A)

Stereo ribbon diagram representation depicting a-helices H1–H20

(red) and b-strands S1–S16 (blue). Also shown in ball and stick rep-

resentation the active site His334 and docked substrates choline and

coenzyme A (CoA). Stereo ball and stick diagrams of the modeled

binding site of (B) choline and (C) CoA.


adopt a similar topology with each other in the absenceof any recognizable amino acid sequence homology

(�11% identity) between them.

The overall surface area of the molecule is 25 000�A2.

Out of the 644 amino acids built, 89 (mostly hydro-

phobic residues) are buried at the interface between the

two domains (�4000�A2). In domain I, residues Ala109,

Ile111, and Glu347 have hydrogen bond interactions with

domain II amino acids Thr555, Met556, Phe559, andCys561. The carbonyl oxygens of Glu347 hydrogen bond

with the side-chain of Thr555 (3.1, 2.8�A) and the main-

chain nitrogen of Met556 (2.8�A). Both residues, Ala109

and Ile111, have hydrogen bond interactions with Cys561

(3.0�A) and Phe559 (2.8�A) main-chain atoms. These in-

teractions are predominantly main-chain and therefore

there is no evolutionary pressure to conserve them be-

tween species (Fig. 2).

3.4. Structural comparison of rChAT with hpCAT,

mCAT, and E2p

The amino acid sequence of rChAT is highly ho-

mologous with ChATs from other species and has 95,

86, and 85% identity with mouse, human, and pig,

respectively (Fig. 2). In addition, the amino acid se-

quence of rChAT has �40% identity compared to

hpCAT and mCAT (Wu et al., 2003; Jogl and Tong,2003) and shares a common structure with a RMS

deviation of 1.30 and 1.25�A, respectively, for approx-

imately 570 Ca atoms. The rChAT structure has two

additional b-strands (S9 and S10) and an additional

helix (H14) compared to the structures of hpCAT and

mCAT. Most of the main-chain structural variation

(3.0–6.0�A) occurs between residues 209–216 and 233–

240 of rChAT compared to hpCAT and mCAT(Fig. 3).

Interestingly, E2p, an enzyme that catalyzes a similar

reaction to that of rChAT, in that it transfers an acetyl

group from acetyl-CoA to an organic substrate, shares

an 8% sequence identity with rChAT and when struc-

turally aligned with domain I of rChAT has an RMS

deviation of 1.87�A for 117 Ca atoms (Mattevi et al.,

1993).

4. Discussion

4.1. Catalytic center

The catalytic center of rChAT is a 16�A long tunnel,

formed between the interface of domains I and II.Chemical modification, inactivation, and mutagenesis

studies have identified the catalytic histidine of rChAT

as His334, which is located at the center of this tunnel

between b-strand S8 and helix H12 in domain I (Fig. 1)

(McGarry and Brown, 1997; Ramsay et al., 2001). This

observation is consistent with sequence homology

comparison with the known catalytic histidine residues

of other acyl group transfer enzymes including His322 inhpCAT (Wu et al., 2003), His343 in mCAT (Jogl and

Tong, 2003), His610 in E2p (Mattevi et al., 1993), and

His195 in chloramphenicol transferase (Leslie et al.,

1988).

In rChAT, His334 assumes an unusual conformational

arrangement (v1 ¼ �146 �; v2 ¼ �49 �) to form a hy-

drogen bond (3.0�A) interaction between the imidazole

nitrogen Nd1 and its main-chain carbonyl oxygen. Inthe crystal structure of hpCAT, mCAT, E2p, and

chloramphenicol transferase, the catalytic histidine also

shows a similar conformation (Jogl and Tong, 2003;

Leslie et al., 1988; Mattevi et al., 1993; Wu et al., 2003).

Interestingly, this conformation of His334 allows the N3

nitrogen to point towards the hydroxyl group of choline

to extract a proton (Fig. 1B).

Fig. 2. Sequence alignment of choline acetyltransferases (ChAT). Shown the amino acid sequence alignment of rat, human, mouse, pig, Drosophila

melanogaster, and C. elegans ChAT. The amino acid numbering and secondary structural elements are based on the structure of rChAT. Strands S1–

S16 are represented by blue arrows while helices H1–H20 are depicted by red spirals. Identical and conserved residues are indicated by red and yellow

blocks, respectively. Residues mutated using site-directed mutagenesis are indicated by purple triangles (Carbini and Hersh, 1993; Cronin, 1997; Wu

and Hersh, 1995). Residues mutated in patients with congenital myasthenic syndrome with episodic apnea (CMS–EA) are shown by green triangles

(Ohno et al., 2001). A blue star indicates the residue, Arg312, that was mutated by site-directed mutagenesis and shown to be mutated in patients with

CMS–EA.


In addition, the catalytic histidine is highly conservedamong other members of the LL-carnitine/choline acyl-

transferase enzyme family (His473 in rat carnitine palmi-

toyltransferase I, His372 in carnitine palmitoyltransferaseII, and His327 in carnitine octanoyltransferase) and

modification of these residues confirmed inactivation of

Fig. 3. Structural comparison of rChAT and hpCAT and mutations in hChAT that cause myasthenic syndrome associated with episodic apnea

(CMS–EA). Coil diagram shows the superimposition of rChAT (red) onto hpCAT (blue). Arrows show structural variation (Ca RMS deviation

>2.0�A), residues 209–216 and 233–241, between rChAT and hpCAT. Shown in ball and stick His334, choline/LL-carnitine, and coenzyme A. Residues

mutated in patients with CMS–EA are shown by green spheres (Ohno et al., 2001). Residues mutated using site-directed mutagenesis are indicated by

purple spheres (Carbini and Hersh, 1993; Cronin, 1997; Wu and Hersh, 1995). A blue sphere indicates the residue, Arg312, that was mutated by site-

directed mutagenesis and shown to be mutated in patients with CMS–EA (refer to text and Fig. 2).


these enzymes (Brown et al., 1994; Morillas et al., 2001).

Positioned in the center of the catalytic tunnel, His334 is

accessible from either entrance of the catalytic tunnel

permitting access of both substrates to the catalytic activesite (Fig. 1A).

A highly conserved glutamate, Glu338, interacts with

His334 and may be important for catalytic activity. The

carbonyl oxygen O1 of Glu338 is hydrogen bonded

(3.7�A) with the N3 of the active site His334 and the O2

of Glu338 hydrogen bonds (2.8�A) with Arg458. Most

likely these interactions contribute to potentiate the

Table 2

Choline and CoA substrate binding sites for rChAT, hpCAT, and mCAT

Atom Residue [distance (

rChAT

Choline/LL-Carnitine binding site

O3 His334 (3.02)

N(CH3)3 Ser548 (3.04)

Tyr562 (4.6)

Val565 (3.16)

O1 —

O2 —

CoA binding site

P3(O9) Lys413 (3.23)

Lys417 (2.72)

a Interactions taken from the crystal structure of hpCAT, PDB No., 1S5b Interactions taken from the crystal structure of mCAT, PDB No., 1ND

catalytic activity of His334 and also stabilize the binding

of choline.

4.2. Molecular docking of choline and CoA at active site

4.2.1. Choline

The docking of choline into the active site of rChAT

indicates five residues, Tyr95, His334, Ser548, Tyr562, and

Val565, that may be involved in its binding (Fig. 1B,

Table 2). The hydroxyl group of choline interacts

with His334 and forms a hydrogen bond (3.0�A). This

�A)]

hpCATa mCATb

His322 (2.76) His434 (2.68)

Ser533 (3.61) Ser552 (3.64)

Phe545 (3.6) Phe545 (3.96)

Val548 (3.83) Val569 (3.86)

Tyr431 (2.55) Tyr452 (2.59)

Thr444 (2.82) Thr465 (2.63)

Lys398 (3.27) Lys419 (3.64)

Lys402 (2.67) Lys423 (3.58)

O (Govindasamy et al., 2004).

F (Jogl and Tong, 2003).


hydrogen bond formation supports the catalytic role ofHis334. Tyr95 is also within hydrogen bonding distance

to the hydroxyl group of choline (2.0�A). The quater-

nary amine head group of choline is positioned close to

the side-chain of Tyr562, separated by 4.6�A, and has

hydrophobic interactions with the side-chain residues

Ser548 and Val565 (3.0�A and 3.2�A, respectively)

(Fig. 1B, Table 2). Interestingly, in other highly con-

served members of the LL-carnitine/choline acyltrans-ferase family, hpCAT, and mCAT, the quaternary

amine of LL-carnitine appears to be positioned close to a

phenylalanine residue, Phe545 and Phe566, respectively.

However, in the case of rChAT the quaternary amine

of choline interacts with a tyrosine residue (Tyr562)

(Jogl and Tong, 2003; Wu et al., 2003). In hpCAT, the

highly conserved residues within carnitine acyltrans-

ferases, Thr444 and Arg497 (Val459 and Asn514 inrChAT) play critical roles by interacting with the car-

boxylate group of LL-carnitine (Govindasamy et al.,

2004; Jogl and Tong, 2003). Since choline does not

have a carboxylate group at this position, these resi-

dues are not conserved in rChAT.

All members of the LL-carnitine/choline acyltransfer-

ase family contain a conserved Ser–Thr–Ser (STS) motif

near the carboxy terminal, Ser548, Thr549, and Ser550 inrChAT (Cronin, 1997). The first serine of this motif,

Ser548, is positioned close to the quaternary amine group

of choline and may play a critical role in transition state

stabilization during catalysis.

4.2.2. CoA

The CoA binding site was modeled on the opposite

side of His334 from the choline binding site (Fig. 1).The strands S11 and S12 of domain II are separated

from each other and create a space that can accom-

modate the pantothenic arm of CoA, which extends

towards the active site. Highly conserved residues in LL-

carnitine/choline acyltransferases surround the

CoA binding site at the C-terminal end of the enzyme

(Table 2).

Residues Lys413 and Lys417 may interact with the 30-phosphate group of CoA and a clustered group of res-

idues, Asp424, Glu447, and Gln551, possibly interact with

the pantothenic arm of CoA (Fig. 1C, Table 2). The

carboxyl oxygens of Asp424 are hydrogen bonded with

Gln551 (3.0�A) and Glu447 (2.6�A). This would imply at

least some effect on the protonation state of at least one

of the acidic residues in the active site.

Previous chemical modification, inactivation, andsite-directed mutagenesis studies have suggested that

Arg452 of rChAT interacts with the 30-phosphate group

of CoA to stabilize CoA binding (Mautner et al., 1981;

Wu and Hersh, 1995). Although substitution of this

residue by an alanine resulted in a 7–12 fold increase in

Km for both CoA and acetyl-CoA as well as an increase

in the kcat value (Wu and Hersh, 1995), based on ob-

servations from the structure of rChAT it appears thatthis residue is too far from the 30-phosphate group of

CoA (�11�A) to form an interaction. Perhaps the mu-

tation of Arg452 with alanine disrupts the overall struc-

ture of the active site or the stability of the enzyme to

cause such a decreased affinity towards the substrates.

4.3. Diseases causing mutations

CMS–EA is an inherited disease that is character-

ized by severe dyspnea, respiratory, and bulbar weak-

ness that can lead to respiratory failure. These

manifestations are precipitated by fever, exertion, and

excitement that can be prevented or alleviated by

acetylcholinesterase inhibitors (Engel et al., 2003).

Previous studies have shown defects in ChAT, acetyl-

cholinesterase, the acetylcholine receptor, and thepostsynaptic molecule RAPSYN as the cause of the

disease (Engel et al., 2003).

An analysis of the ChAT encoding gene from five

patients with CMS–EA revealed recessive mutations,

one (523insCC) null mutation, three mutations

(Ile305Thr, Arg420Cys, and Glu441Lys) that reduce

ChAT expression, one mutant (Glu441Lys) devoid of

catalytic activity and eight mutants (Leu210Pro,Pro211Ala, Ile305Thr, Arg420Cys, Arg482Gly, Ser498Leu,

Val506Leu, and Arg560His) that have decreased catalytic

efficiencies towards CoA (Figs. 2 and 3) (Ohno et al.,

2001). Additional mutational analysis of the ChAT

encoding gene from three patients with CMS–EA from

two Turkish families shows an additional mutation

(Ile336Thr) and studies from five patients from three

independent families shows three further mutations(Val194Leu, Arg548Stop, and Ser694Cys) (Maselli et al.,

2003; Schmidt et al., 2003). Fig. 3 shows that most of

these residues are located in the periphery of the

structure, away from the active site tunnel, and perhaps

are important for stability or maintaining the confor-

mation of the active site.

The most severe mutation reported, Glu441Lys,

drastically reduces ChAT expression in COS cells, 29%compared to wild type, and the mutant form of the

enzyme is devoid of catalytic activity (Ohno et al., 2001).

As shown in Fig. 2, this conserved residue corresponds

to Glu333 in rChAT and is located in strand S8, adjacent

to the catalytic histidine. Although this residue points in

the opposite direction of the active site and appears to

not be involved directly in the catalytic mechanism of

the enzyme, mutation to a lysine could introduce inter-actions with a nearby arginine, Arg250 (4.0�A). These

interactions could disrupt the active site or the orienta-

tion of the active site histidine and thus eliminate cata-

lytic activity.

Another interesting mutation is Arg560His. This mu-

tation causes a decreased affinity towards both acetyl-

CoA and choline (Ohno et al., 2001). Arg560 is abso-


lutely conserved and corresponds to Arg452 in rChAT(Fig. 2). As previously discussed, chemical modification,

inactivation, and site-directed mutagenesis studies have

shown that this residue interacts with the 30-phosphategroup of CoA (Mautner et al., 1981; Wu and Hersh,

1995). However, based on the structure, Arg452 does not

interact with either substrate. Therefore, it is quite per-

plexing that mutation of this residue can impair catalytic

activity to such a degree that it causes a disease state.Based on the structure, it appears that a histidine lo-

cated at position 452, could interact with Gln164

(�2.5�A), which could disrupt the conformation of the

active site histidine. A histidine residue at position 452

could also interact with Glu338 (�4.0�A), which, as pre-

viously discussed, forms a hydrogen bond interaction

with the active site histidine.

The other mutations, Leu102Pro, Pro103Ala, Ile197Thr,Arg312Cys, Arg374Gly, Ser390Leu, and Val398Leu, have

been shown to cause a decrease in the affinity for acetyl-

CoA and therefore decrease catalytic efficiency when

compared to wild type (Ohno et al., 2001).

4.4. Temperature sensitive mutants

The structure of rChAT can also be used to under-stand the temperature sensitive mutations in Drosophila

that were generated to determine the role of the neuro-

transmitter acetylcholine in development and mediating

behavioral function in the brain (Greenspan et al.,

1980). These mutations, Chats1 and Chats2, show a

conditional temperature sensitive phenotype in which at

increased temperatures, 30 �C, ChAT activity decreases.

Drosophila expressing either mutation become paralyzedand subsequently die when incubated at 30 �C (Green-

span et al., 1980). At the higher temperatures it was

shown that the mutant phenotype appears more rapidly

with the Chats2 mutant, indicating that the Chats2 mu-

tation is more severe than the Chats1 (Greenspan et al.,

1980).

Recently, these mutations have been identified as

Met403Lys for Chats1, which corresponds to Leu318 inrChAT, and Arg397His for Chats2, which corresponds

to Arg312 in rChAT (Fig. 2) (Wang et al., 1999).

Arg312 is absolutely conserved within all known

ChATs while Leu318 is not conserved. In fact, this

residue is a leucine residue in rat, human, mouse, and

pig, an isoleucine in C. elegans, and a methionine in

the Drosophila ChAT.

Both of these residues are close to the active sitehistidine. The replacement of Leu318 by lysine appears

to affect the overall stability of the enzyme since it

appears that this mutation does not introduce delete-

rious interactions with surrounding residues. In con-

trast, substitution of Arg312 by histidine may change

the choline binding site. It appears that a histidine

residue located at position 312 may interact with

Glu333, which is located adjacent to the active sitehistidine. As previously discussed, this residue faces the

opposite direction of the active site. However, it may

be critical in formation of the choline binding pocket,

such that an unfavorable interaction will disrupt cho-

line binding.

5. Conclusion

The structure of rChAT has been determined to a

1.55�A resolution and exhibits conserved structural fea-

tures found in hpCAT and mCAT (Wu et al., 2003; Jogl

and Tong, 2003). RChAT is a monomeric protein con-taining two domains that interconnect to form an active

site tunnel. Within the active site the catalytic residue

His334 is located at the center of the tunnel, suggesting a

common catalytic mechanism for the entire LL-carnitine/

choline acyltransferase family. The substrates, choline

and acetyl-CoA, have been modeled into the active site,

based on the structure of mCAT, and reveal several

residues that may be involved in substrate recognitionand ligand interactions and provide insight into the

molecular basis of disease causing mutations.

Acknowledgments

This research is supported in part by NIH Grant

GM58197 (D.H.W) and the University of Florida,

College of Medicine start-up funds (R.M.). Thomas

Kukar and Brenda Pedersen are supported by a Uni-

versity of Florida Alumni Fellowship. Figs. 1 and 3 were

generated with the program Bobscript (Esnouf, 1997) in

combination with RASTER-3D (Merritt and Bacon,1997). The alignment in Fig. 2 was generated using

ClustalW (Thompson et al., 1994) and secondary

structure was added using ESPript 2.1 (Gouet et al.,

1999).

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Structural insights and functional implications of choline acetyltransferase

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