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
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.
L. Govindasamy et al. / Journal of Structural Biology 148 (2004) 226–235 229
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.
230 L. Govindasamy et al. / Journal of Structural Biology 148 (2004) 226–235
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).
L. Govindasamy et al. / Journal of Structural Biology 148 (2004) 226–235 231
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).
232 L. Govindasamy et al. / Journal of Structural Biology 148 (2004) 226–235
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-
L. Govindasamy et al. / Journal of Structural Biology 148 (2004) 226–235 233
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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