Molecular identification of N-acetylaspartylglutamate synthase and β-citrylglutamate synthase

Article abstract of DOI:10.1074/jbc.M110.152629

The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and L-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and β-citrylglutamate.

Full text of DOI:10.1074/jbc.M110.152629

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 39, pp. 29826–29833, September 24, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Molecular Identification of N-Acetylaspartylglutamate
□
S
Synthase and ␤-Citrylglutamate Synthase*
Received for publication, June 9, 2010, and in revised form, July 14, 2010 Published, JBC Papers in Press, July 24, 2010, DOI 10.1074/jbc.M110.152629
Franc¸ois Collard‡1, Vincent Stroobant§, Pedro Lamosa¶, Coco N. Kapandaʈ, Didier M. Lambertʈ, Giulio G. Muccioliʈ,
Jacques H. Poupaertʈ, Fred Opperdoes**, and Emile Van Schaftingen‡2
From the ‡Laboratory of Physiological Chemistry, the §Ludwig Institute for Cancer Research, and the **Research Unit for Tropical
Diseases, de Duve Institute and Universite´ Catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium, the ʈUnite´ de
Chimie Pharmaceutique et de Radiopharmacie and Bioanalysis and Pharmacology of Bioactive Lipids Laboratory, Louvain Drug
Research Institute, Universite´ Catholique de Louvain, Av. E. Mounier 72, CHAM7230, B-1200 Brussels, Belgium, and the ¶Instituto de
Tecnologia Química e Biolo´gica, Universidade Nova de Lisboa, Centro de Ressonaˆncia Magne´tica Anto´nio Xavier, Av. da Repu´blica,
Estac¸a˜o Agrono´mica Nacional, 2780-157 Oeiras, Portugal
The purpose of the present work was to determine the identity
of the enzymes that synthesize N-acetylaspartylglutamate
(NAAG), the most abundant dipeptide present in vertebrate cen-
tral nervous system (CNS), and ␤-citrylglutamate, a structural ana-
logue of NAAG present in testis and immature brain. Previous evi-
dence suggests that NAAG is not synthesized on ribosomes but
presumably is synthesized by a ligase. As attempts to detect this
ligase in brain extracts failed, we searched the mammalian
genomes for putative enzymes that could catalyze this type of reac-
tion. Mammalian genomes were found to encode two putative
ligases homologous to Escherichia coli RIMK, which ligates gluta-
mates to the C terminus of ribosomal protein S6. One of them,
named RIMKLA, is almost exclusively expressed in the CNS,
whereas RIMKLB, which shares 65% sequence identity with
RIMKLA, is expressed in CNS and testis. Both proteins were
expressed in bacteria or HEK293T cells and purified. RIMKLA
catalyzed the ATP-dependent synthesis of N-acetylaspartylglu-
tamate from N-acetylaspartate and L-glutamate. RIMKLB cata-
lyzed this reaction as well as the synthesis of ␤-citrylglutamate.
The nature of the reaction products was confirmed by mass
spectrometry and NMR. RIMKLA was shown to produce stoi-
chiometric amounts of NAAG and ADP, in agreement with its
belonging to the ATP-grasp family of ligases. The molecular
identification of these two enzymes will facilitate progress in the
understanding of the function of NAAG and ␤-citrylglutamate.
most particularly in neurons of the anterior horn (1). NAAG
can be released from neurons upon calcium depolarization
(reviewed in Ref. 1) and is a substrate for two glial peptidases,
glutamate carboxypeptidase-II (2) and, to a lower extent, gluta-
mate carboxypeptidase-III (3), which are anchored to the
plasma membrane with their catalytic site oriented toward the
outside of the cell. NAAG long has been thought to be a neuro-
transmitter able to bind to the metabotropic glutamate recep-
tors mGluR3 (4). Two recent reports suggest, however, that the
effects of NAAG as a neurotransmitter are due to a 0.5% gluta-
mate contamination present in commercial NAAG (5, 6). The
function of NAAG is, therefore, presently unknown.
␤-Citrylglutamate (BCG), which is structurally close to NAAG,
is less well characterized. It was first identified in newborn rat
brain, where its concentration reaches 0.5–1 ␮mol/g at birth and
then decreases with age (7). BCG also was detected in kidneys and
heart and to a much lower extent in intestine, spinal cord, and lung
of newborn rats. The content of BCG in all organs decreased with
age to the noticeable exception of testes, where its concentration
increases during sexual maturation and remains constant in adult-
hood (8, 9). BCG is present not only in the testes of mammals but
also in those of amphibians and fishes. There is evidence in germi-
nal cells for a role in spermatogenesis (9). BCG recently has been
proposed to be an iron chelator (10).
Little is known regarding the synthesis of NAAG and BCG.
NAAG is synthesized in cells even in the presence of protein syn-
thesis inhibitors, from N-acetylaspartate (NAA), suggesting that it
is formed by a ligase and not on ribosomes (11, 12). The finding
that no NAAG is synthesized in a patient with a defect in aspartate
N-acetyltransferase (NAT8L) indicates that NAAG is indeed syn-
thesized from NAA and glutamate (13, 14). The synthesis of ␤-cit-
rylglutamate by tissues has not been studied. Progression in our
understanding of the function of NAAG and BCG would be
greatly facilitated by molecular identification of the enzyme(s) that
make(s) these two compounds. Our recent success in the identifi-
cation of aspartate and cysteinyl-S-conjugate N-acetyltransferases
by a database search approach led us to attempt the identification
of these (this) enzyme(s) with a similar strategy (14, 15).
N-Acetylaspartylglutamate (NAAG),3 the most abundant di-
peptide in the CNS, is present in brain and in the spinal cord,
* This work was supported by grants from the Interuniversity Attraction Poles
Program-BelgianSciencePolicy(NetworksP6/05andP6/28),bytheDe´sordres
Inflammatoires dans les Affections Neurologiques center of excellence
program of the Re´gion Wallonne, and by a grant from Asco Industries (to
E. V. S.). The NMR spectrometers are part of the National NMR Network (REDE/
1517/RMN/2005), supported by “Programa Operacional Cieˆncia e Inovac¸a˜o
2010” and Fundac¸a˜o para a Cieˆncia e a Tecnologia, Portugal.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Tables S1 and S2.
1 Recipient of a Charge´s de Recherche mandate of the Fonds National de la
Recherche Scientifique.
2 To whom correspondence should be addressed. Tel.: 003227647564; Fax:
003227647598; E-mail: emile.vanschaftingen@uclouvain.be.
3 The abbreviations used are: NAAG, N-acetylaspartylglutamate; BCG, ␤-cit-
MATERIALS AND METHODS
Cloning and Preparation of Expression Vectors—The DNA
rylglutamate; HMBC, heteronuclear multiple bond connectivity spectrum. sequences of RIMKLA and RIMKLB (GenBankTM accession
29826 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 39•SEPTEMBER 24, 2010

Molecular Identification of NAAG and BCG Synthases
nos. NM_177572 and NM_027664) were PCR-amplified from Vivaspin 15 concentration unit (Sartorius), and applied onto a
mouse brain cDNA using 5Ј primers (RIMKLA, ttg agg tac cat S-200 gel filtration column (GE Healthcare) equilibrated with
atg tgc gcg cag gtc tgg; RIMKLB, tt cgg tac cat atg tgt agc tca gtg buffer C (25 mM Hepes, pH 7.1, 200 mM NaCl, 1 mM DTT, 1
act gg) containing KpnI and NdeI restriction sites (in boldface ␮g/ml leupeptin and antipain), and fractions were collected.
type) and 3Ј primers (RIMKLA, gcg gat cct taa tgc tgt aac cag The purification of His-tagged RIMKLA and RIMKLB was per-
gct tgg g; RIMKLB, gcg gat cct cac tcc acc agg agt ttg att tc) formed as for RIMKLA, except that the Q-Sepharose purifica-
containing a BamHI site and inserted in the pEF6Myc-His tion step was replaced by a purification on a HisTrap column
eukaryotic expression vector at the KpnI-BamHI sites. To cre- (5-ml, GE Healthcare) performed as described in Ref. 16.
ate fusion proteins with the polyhistidine tag at the C terminus,
RIMKLB was purified starting from transfected HEK293T
the stop codons were removed by mutagenesis. The resulting cells (30 dishes of 60 cm2). Cells were collected and resus-
pEF6Myc-His constructs were used to transfect HEK293T cells pended in buffer A, thawed, lysed by vortex-mixing, and centri-
(results not shown). To express the His-tagged RIMKLA/B pro- fuged for 30 min at 20,000 ϫ g. The supernatant contained the
teins in bacteria, the DNA fragment corresponding to the ␤-citrylglutamate synthase activity (Ͼ90%) and was used for
RIMKLA/B open reading frames in fusion with the C terminus the purification. The purification procedure was identical to
polyhistidine tag were excised from the pEF6MycHis con- the purification of RIMKLA (see above).
structs by restriction with NdeI and MssE (blunt) and inserted
Western Blots—Western blots were performed as described
into a (5Ј)NdeI-(3Ј)blunt pET3a vector. A BL21(DE3)pLysS previously (17) with serum of rabbits immunized against mouse
strain was used for the expression of His-tagged RIMKLA, and RIMKLA or RIMKLB peptides coupled to KLH (Eurogentec)
a BL21 GroE strain was used for His-tagged RIMKLB. Bacterial diluted 1:2000 in PBS containing 1% (w/v) BSA. After washing,
expression of the non-His-tagged RIMKLA and RIMKLB was membranes were probed for 1 h at room temperature with per-
performed using the pET3a expression plasmid. PCR-amplified oxidase-conjugated anti-rabbit Ig antibody (Sigma-Aldrich).
RIMKLA and RIMKLB were inserted in the pET3a expression Detection was performed using the ECL Western blot analysis
vector at the NdeI-BamHI sites. A BL21(DE3)pLysS strain was system (GE Healthcare) according to the manufacturer’s
used for the expression of RIMKLA, and a BL21 GroE strain was protocol. Peptide sequences used for RIMKLA antibodies pro-
used for RIMKLB. All expression vectors were checked by duction were CGLQLSQKPLTTFPD and ILPGLASPREKNEP-
sequencing.
NGC, and sequences for RIMKLB were CDPESTTEREML-
Expression in Bacteria—1 liter of LB medium was inoculated TKLP and AGRLTRRMSLLS (see Fig. 1).
with 25 ml of preculture in the presence of 200 ␮g/l ampicillin
Enzymatic Assays—RIMKLA and RIMKLB were assayed
(BL21(DE3)pLysS and BL21 GroE strains) and 25 ␮g/l chloram- radiochemically in a mixture (final volume, 200 ␮l) comprising,
phenicol (BL21(DE3)pLysS strain only) and incubated at 37 °C unless otherwise stated, 25 mM Tris, pH 8.0, 5 mM DTT, 5 mM
under vigorous shaking until A600 reached 0.6. The flasks were MgATP, 5 mM MgCl2, 1 mM EGTA, 50,000 cpm L-[U-14C]glu-
then cooled on ice for 15 min. Isopropyl 1-thio-␤-D-galactopy- tamate (GE Healthcare), L-glutamate (in a concentration rang-
ranoside was added to a final concentration of 0.4 mM, and the ing from 0.05 to 1 mM), 1 mg/ml BSA, and 5 mM NAA or citrate.
culture was pursued for 24 h at 16 °C. Bacterial extracts were After 30 min at 30 °C, the reaction was stopped by a 5-min
prepared as described previously and centrifuged for 40 min at incubation at 80 °C and centrifuged for 20 min at 15,000 ϫ g,
20,000 ϫ g (16).
and the supernatant was diluted in 0.8 ml of 5 mM Hepes, pH
Expression in HEK293T Cells—HEK293T cells were cultured 7.1. The sample was applied onto a 1 ml Dowex AG1-X8 col-
Ϫ
and transfected essentially as described by Ref. 17 using the umn (Cl form, 100–200 mesh, Acros Organics) prepared in a
jetPEITM procedure. After 48 h at 37 °C, the cells were washed Pasteur capillary pipette. The latter was washed with 2 ml of 5
once with 5 ml of PBS. They were scraped into 0.8 ml of buffer mM Hepes, followed by 5 ml of 150 mM NaCl in the same buffer
A (25 mM Hepes, pH 7.1, 1 mM ␣-toluenesulfonyl fluoride, 1 to elute unreacted glutamate and 2 ϫ 2 ml of 500 mM NaCl to
␮g/ml leupeptin and antipain), frozen in liquid nitrogen, elute NAAG or ␤-citrylglutamate. Radioactivity was counted in
thawed, and lysed by vortex-mixing.
the presence of Ultima Gold (PerkinElmer Life Sciences) in a
Purification—RIMKLA was purified on DEAE-Sepharose, liquid scintillation counter.
Q-Sepharose, and gel filtration. 50 ml of a bacterial extract
NMR and MS Characterization of NAAG Formed Enzymati-
supernatant (corresponding to 2 liters of culture) were diluted cally by RIMKLA—50 milliunits of purified His-tagged
in 150 ml of buffer A (25 mM Hepes, pH 7.1, 1 mM TSF, 1 ␮g/ml RIMKLA was added to a 100-ml solution containing 25 mM Tris,
leupeptin and antipain) and loaded onto a 25-ml DEAE column pH 8,0, 5 mM NAA, 5 mM MgATP, 5 mM MgCl2, 10 mM DTT, 0.2
(GE Healthcare) in a Bio-Rad FPLC. The column was washed mg/ml bovine serum albumin, 5 mM glutamic acid, and 500,000
with 75 ml buffer A, a linear 250 ml gradient (0 to 0.5 M NaCl in cpm L-[U-14C]glutamate. The reaction mixture was incubated
buffer A) was applied, and fractions were collected. NAAG syn- overnight at 30 °C under stirring and then heated for 10 min at
thase activity was assayed, and active fractions were pooled, 80 °C. Themixturewascentrifuged30minat18,000 ϫgtoremove
diluted with 4 volumes of buffer B (25 mM Tris, pH 8.0, 1 mM proteins, and the supernatant was treated with 2% (w/v) activated
DTT, 1 ␮g/ml leupeptin and antipain), and loaded onto a 20-ml charcoal to remove nucleotides. The charcoal was filtered, and the
Ϫ
Q-Sepharose column. The column was washed with 50 ml of filtrate was loaded onto a 25-ml AG1-X8 Dowex column (Cl
buffer B, a linear 250-ml gradient (0 to 0.5 M NaCl in buffer B) form). The column was washed with 100 ml of water, a linear
was applied, and fractions were collected. Fractions containing gradient of NaCl was applied (0 to 1 M NaCl in 300 ml), and frac-
NAAG synthase activity were pooled, concentrated to 2 ml on a tions (5 ml) were collected. Fractions containing radioactivity cor-
SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
JOURNAL OF BIOLOGICAL CHEMISTRY 29827

Molecular Identification of NAAG and BCG Synthases
scopic analyses. All spectra were
acquired on a Bruker AVANCE III
800 spectrometer (Bruker, Rhein-
stetten, Germany) working at a pro-
ton operating frequency of 800.33
MHz, equipped with a three chan-
nel 5-mm inverse detection probe
head with pulse field gradients along
the Z axis. Spectra were run at 25 °C
using standard Bruker pulse pro-
1
grams. H and 13C chemical shifts
are referenced to 3-(trimethylsilyl)-
1
propane sulfonic acid. The H-13C
heteronuclear multiple bond con-
nectivity spectrum (HMBC) was
modified to include a water pre-
saturation pulse during the relax-
ation delay and a carbon decou-
pling GARP4 sequence (18) during
the acquisition time. A delay of 71
ms was used for the evolution of the
J
H,C long range couplings.
RESULTS
Identification of Putative Mam-
malian NAAG Synthetases—As at-
tempts to detect the activity of
the enzyme synthesizing NAAG in
mouse brain extracts failed, we
decided to use a database search
approach to identify this enzyme.
Previous reports indicated that
FIGURE 1. Sequence alignment of mouse RIMKLA and RIMKLB with E. coli RIMK and E. coli glutathione
synthase. The following sequences are shown: mouse RIMKLA (MmusA, NP_808240), mouse RIMKLB (MmusB,
NP_081940), E. coli RIMK (EcRIM, NP_415373), E. coli glutathione synthetase (EcGlS, NP_417422, Protein Data
Bank code 1GSH). Strictly conserved residues are indicated in boldface type. Asterisks indicate residues of the
catalytic site involved in the binding of glutathione synthase substrates (see “Discussion”). The peptides used
to prepare anti-RIMKLA and anti-RIMKLB antibodies are underlined.
NAAG is synthesized from N-acetylaspartate and glutamate in
the presence of ATP (12, 19, 20). This suggests that the synthe-
sis of NAAG is catalyzed by a ligase using ATP. Many ligases
catalyzing amide bond formation belong to the “ATP-grasp”
clan. Searching in the Pfam database (21) for mouse or human
enzymes of unknown function and belonging to this clan indi-
cated that two of them, RIMKLA and RIMKLB, belonged to a
family (the RIMK family) that comprises three enzymes ligating
the ␣-amino group of glutamate to the carboxylic group of an
acceptor. The prototypic enzyme RIMK adds a glutamate at the
C terminus of ribosomal protein S6 (22), whereas two other
enzymes participate in the synthesis of glutamate containing
cofactors, namely coenzyme F420 and tetrahydrosarcinapterin
(23). These findings suggested that RIMKLA and RIMKLB also
served to ligate free glutamate to (an) acceptor(s).
responding to NAAG were pooled, concentrated to 2 ml in a
lyophilizer, and loaded onto a Bio-Gel P2 column (Bio-Rad; 50
cm ϫ 1.0 cm) equilibrated with water to separate NaCl from
NAAG. Desalted fractions containing NAAG were evaporated
and analyzed by NMR and MS. MS analysis was performed on a
LCQ Deca XP ion-trap spectrometer equipped with an electro-
spray ionization source (ThermoFinnigan, San Jose, CA). The
LCQ was operated in positive mode under manual control in the
Tune Plus view with default parameters and active automatic gain
control. MS/MS analysis was done to confirm the structure of the
precursor ions using low energy collision-induced dissociation
with a relative collision energy of 25%. For NMR analysis, the sam-
ple was dissolved in 500 ␮l of H2O/D2O (9:1) and transferred to a
5-mm NMR tube. Spectra were recorded on a Bruker Avance 400
MHz UltrashieldTM spectrometer.
NMR and MS Characterization of ␤-Citrylglutamic Formed
RIMKLA and RIMKLB are Ϸ 390 amino acid proteins that
Enzymatically by RIMKLB—␤-Citrylglutamic acid was en- are predicted to be soluble. They share ϳ85% sequence identity
zymatically prepared using His-tagged RIMKLB (50 mU), among each other, 30% identity with E. coli RIMK, and 25%
and the same reaction mixture as described above except identity with E. coli glutathione synthase (Fig. 1). Compared
that NAA was replaced by citrate. For the synthesis of 13C- with the latter two sequences, RIMKLA and RIMKLB present
citrate-labeled citrylglutamate, 13C-citrate (CortecNet) was C-terminal extensions of ϳ70 residues that show a much lower
used, and the final volume was reduced to 2 ml. Purification and degree of conservation. BioGPS expression data indicate that
MS analysis of ␤-citrylglutamate was performed as for NAAG. RIMKLA is mostly expressed in the CNS, most particularly in
NMR analysis was performed on purified 13C citrate-labeled the spinal cord and in dorsal root ganglions, but also in the
citrylglutamate. The sample was dissolved in 500 ␮l of H2O/ retina (GeneAtlas MOE430; probe 1555378). RIMKLB is
D2O (9:1) and transferred to a 5-mm NMR tube for spectro- expressed in testis, placenta, and various regions of the CNS
29828 JOURNAL OF BIOLOGICAL CHEMISTRY
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Molecular Identification of NAAG and BCG Synthases
(probe 1435532). These findings suggested that RIMKLA and
RIMKLB could be involved in the synthesis of NAAG and ␤-cit-
rylglutamate, respectively.
Characterization of RIMKLA—To check the function of
RIMKLA, we produced it in Escherichia coli either unmodified
or as a fusion protein with a poly-His tag at the C terminus.
Extracts of cells expressing these recombinant proteins dis-
played NAAG synthase activity, as indicated by the conversion
of radiolabeled glutamate to a more anionic product in the pres-
ence of NAA and ATP. No conversion was observed in the
absence of either of these compounds or when a control bacte-
rial extract was used. Western blotting using an anti-RIMKLA
peptide antibody and an anti-His tag antibody indicated that
the His-tagged protein was partially (Ͼ50%) proteolysed,
whereas this was not the case for the nontagged protein. Both
the tagged and the nontagged proteins were purified as de-
scribed in “Materials and Methods,” to purities of ϳ60 and 30%
as estimated from Coomassie Blue stained SDS-PAGE gels
(data not shown). Both preparations were active and showed
similar kinetic properties (see below), despite the fact that the
purified His-tagged protein was partially proteolysed (data not
shown). Fig. 2A shows the elution profile of the nontagged pro-
tein from a Sephacryl S-200 column. The activity was present in
the flow-through fractions and coeluted with a band with the
expected size (43 kDa) that reacted with antipeptide antibodies
in Western blotting. A comparison of its elution profile with
that of molecular mass standards indicated that the apparent
molecular mass of the holoenzyme was Ͼ250 kDa, i.e. that the
enzyme comprises at least six monomers. Similar results were
obtained with the His-tagged protein.
To confirm the identity of the product made by RIMKLA
from NAA and glutamate in the presence of ATP, His-tagged
RIMKLA was allowed to synthesize ϳ1 ␮mol of product, which
was purified by anion exchange chromatography and gel filtra-
tion. Mass spectrometry analysis of this product in the positive
mode indicated the presence of a major peak of m/z 305 corre-
sponding to NAAG. Fragmentation revealed the appearance of
a major fragment with m/z 148, corresponding to protonated
glutamic acid, indicating the loss of the NAA moiety. This
information indicated that the synthesized molecule contained
NAA and glutamate but did not disclose whether the amide
bond involved the ␣- or ␤-carboxylic group of NAA. To deter-
mine the structure of the synthesized compound, its 13C NMR
spectrum was recorded and compared with those of commer-
cial ␣- and ␤-NAAG. The spectrum of the enzymatically syn-
thesized NAAG matched that of ␣-NAAG and was distinctly
different from that of ␤-NAAG (supplemental Table S1). These
data led to the conclusion that RIMKLA indeed synthesizes
␣-NAAG, the physiological form of NAAG that is present in
vertebrate CNS.
FIGURE 2. Purification of recombinant untagged RIMKLA (A) and RIMKLB
(B) by gel filtration on Sepharose S-200. Mouse RIMKLA was produced in
E. coli and purified by chromatography on DEAE-Sepharose and Q-Sepha-
rose. The most active fractions of the latter column were applied on a Sepha-
rose S-200 column. NAAG synthase activity (F) was measured in the fractions.
RIMKLA was detected by Western blotting using anti-RIMKLA peptide anti-
bodies. Mouse RIMKLB was produced in HEK cells and purified as RIMKLA
before being loaded on the gel filtration column. The ␤-citrylglutamate syn-
thase activity (F) of this enzyme was measured in fractions. mU, milliunits
absorbance at 280 nm.
The partially purified untagged enzyme was used to study the
kinetic properties of RIMKLA. As expected, the activity of this
enzyme was dependent on the simultaneous presence of NAA,
glutamate, and ATP. No activity was observed when NAA was
replaced by aspartate. As shown in Fig. 4, the pH curve showed
an optimum at ϳ8. The enzyme was markedly stimulated by
dithiothreitol, which increased the activity by ϳ5-fold (data not
shown). Dithiothreitol was therefore added to all enzymatic
assays. The Km for NAA, glutamate and MgATP were 1.48 mM,
0.88 mM, and 0.065 mM, respectively (Table 1). The Vmax
amounted to 1.09 ␮mol/min/mg protein, corresponding to a
As enzymes of the ATP-grasp family catalyze the synthesis of
covalent bonds with concomitant hydrolysis of ATP to ADP
and inorganic phosphate, we checked the identity of the nucle-
otide released when RIMKLA synthesizes NAAG. The enzyme
was found to convert ATP to ADP in stoichiometric amounts
with respect to the formation of NAAG. No AMP was formed
(Fig. 3).
k
cat of Ϸ 2.6/s assuming that the purity of the enzyme was Ϸ
30%. The enzyme also catalyzed the synthesis of ␤-citrylgluta-
mate, with an activity that was ϳ75-fold lower than its NAAG
SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
JOURNAL OF BIOLOGICAL CHEMISTRY 29829

Molecular Identification of NAAG and BCG Synthases
TABLE 1
Kinetic parameters of RIMKLA and RIMKLB
Values are means of three measurements using two and three independent prepa-
rations of RIMKLA and RIMKLB, respectively. Activities were measured as
described under “Materials and Methods” at pH 8.0. The Vmax calculation for
RIMKLA takes into consideration the 30% purity of the preparation. In the case of
RIMKLB, the exact purity of the enzyme could not be estimated precisely enough,
and it was therefore not integrated in the calculation of the Vmax
.
RIMKLA
Vmax
RIMKLB
Vmax
Km
Km
mM
␮mol/min/mg
mM
nmol/min/mg
N-Acetyl aspartate
Citrate
1.48
0.87
0.88
0.065
1.088
0.014
1.014
0.778
4.59
2.35
2.83
2.50
2.30
1.24
Glutamate
ATP
0.73
0.0096
FIGURE 3. Stoichiometry of the formation of NAAG and ADP by RIMKLA.
PurifiedRIMKLA(10.5␮g)wasincubatedat30 °Cinafinalvolumeof1mlof25
mM Tris, pH 8.0, 5 mM DTT, 1 mM MgATP, 5 mM MgCl2, 1 mM EGTA, and 5 mM
glutamate as well as 250,000 cpm of L-[U-14C]glutamate, 1 mg/ml BSA, and 5
mM NAA. At the indicated times, an aliquot (200 ␮l) was taken; 100 ␮l of the
reaction mixture were loaded onto a 1-ml Dowex AG1-X8 (ClϪ form) to assay
radiochemically glutamate consumption and NAAG formation (see “Material
and Methods”), and 100 ␮l were used to measure nucleotides by high pres-
sure liquid chromatography as described in Ref. 34.
chromatography on DEAE-Sepharose and His-trap columns
(data not shown). As for RIMKLA, this enzyme preparation was
partially proteolysed. Nontagged RIMKLB produced by trans-
fection of HEK cells was soluble. It was partially purified by
chromatography on DEAE-Sepharose, Q-Sepharose, and gel
filtration on Sephacryl S-200. Western blotting indicated that it
had the expected size (42 kDa) and was not proteolysed (Fig.
2B). It was, however, much less pure than the His-tagged pro-
tein prepared from bacterial extracts.
The nonproteolysed preparation of RIMKLB produced in
HEK cells was used to study the kinetic properties. Remarkably,
this enzyme catalyzed the synthesis of both NAAG and ␤-cit-
rylglutamate with similar Vmax (Table 1), the Km being ϳ3-fold
lower for citrate than for NAA. Similar to RIMKLA, the activity
of RIMKLB was markedly stimulated (up to 5-fold) by DTT
(data not shown), which was therefore included in all assays and
purification buffers. The pH curves indicated that the rates
for the two reactions were similar between pH 7 and 8, but
that the ␤-citrylglutamate synthetase was severalfold higher
than the NAAG synthetase activity at more alkaline pH (Fig.
4B). The more purified, but proteolysed preparation also cat-
alyzed the synthesis of NAAG and ␤-citrylglutamate at
nearly equal rates. From the Vmax (1.4 and 0.7 ␮mol/min/mg
protein), kcat values of 3 and 1.5/s were calculated for the two
activities, respectively.
To ascertain its identity, the product made by RIMKLB from
citrate and glutamate was purified by anion exchange chroma-
tography and gel filtration. Its MS analysis disclosed a major
cation with m/z 322, as expected for ␤-citrylglutamate (data not
shown). NMR analysis was performed on a compound formed
from uniformly labeled 13C-citrate (Fig. 5). The proton spec-
trum showed six major signals, five corresponding to the reso-
nances of the glutamate moiety and a sixth one with a complex
coupling pattern corresponding to the proton frequencies of
FIGURE 4. pH curve of the NAAG and citrylglutamate synthase activities
of RIMKLA (A) and RIMKLB (B). Purified RIMKLA and RIMKLB activities were
measured as described under “Materials and Methods” using NAA (open sym-
bols) or citrate (closed symbols) as substrate at the indicated pH values. The
buffers used were MES (E), Hepes (ꢀ), and Tris (‚).
synthase activity (Table 1). Similar Km values and pH activity the CH2 groups of citrate, which appear as an AB system split by
dependence were observed for the His-tagged (partially proteo- various JH,C couplings arising from the uniform 13C labeling of
lysed) enzyme (data not shown).
this moiety (Fig. 5). The carbon spectrum presents four major
Characterization of RIMKLB—Recombinant RIMKLB was signals, which were assigned based on the carbon-carbon cou-
much more difficult to produce in soluble form than RIMKLA. plings and chemical shifts. This assignment was further con-
Unmodified RIMKLB was exclusively present in pellets firmed by the acquisition of a carbon-carbon correlation spec-
of centrifuged (at 12,000 ϫ g for 45 min) bacterial extracts, troscopy (COSY) NMR spectrum. The 1H/13C-HMBC (Fig. 5)
as indicated by Western blotting with antipeptide anti- allowed us to firmly establish the amide bond between the NH
bodies. The situation was somewhat better with His-tagged group of glutamate and the carboxyl group at position 4 of
RIMKLB, Ϸ 10% of which was recovered in the supernatant. citrate. In fact, there are clear connectivities between the NH
This form of enzyme could be purified to Ϸ 20% purity by group and carbons at position 4 and 3 of citrate, but not to
29830 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 39•SEPTEMBER 24, 2010

Molecular Identification of NAAG and BCG Synthases
FIGURE 5. 1H/13C-HMBC spectrum of ␤-citrylglutamate formed by
RIMKLB. 13C-Citrate-labeled citrylglutamate was synthesized enzymatically
with RIMKLB and purified as described under “Materials and Methods.” All
spectra were acquired on a Bruker AVANCE III 800 spectrometer.
FIGURE 6. Phylogenetic tree of RIMKLA and RIMKLB. Protein sequences
were obtained form the translated GenBankTM nucleotide database (http://
www.ncbi.nlm.nih.gov/guide) and aligned with each other using ClustalX
(version 2.0) (35). MrBayes (version 3.1.2) (36) was used for the inference of a
phylogenetic tree. The best results were obtained with the WAG (Whelan and
Goldman) model of protein evolution, with a discrete ␥ rate variation model
withfourratecategoriesandaproportionofinvariantresiduesequalto0. The
program was run for 50,000 generations, with a burnin proportion of 0.4.
Supportvaluesforthenodesareindicatedattherespectivebranchingpoints.
The horizontal bar represents 20 accepted mutations per 100 residues. The
E. coli and Methanococcus jannaschii sequences were used as the outgroup.
Homo sapiens A (RIMKLA), NP_775913; H. sapiens (RIMKLB), NP_065785; Rat-
tus norvegicus A, XP_002729492; R. norvegicus B, XP_342750; Mus musculus, A
NP_808240; M. musculus B, NP_081940; Canis familiaris A, XP_539562, C. fa-
miliaris B, XP_543825; Monodelphis domestica A, XP_001367054; M. domes-
tica B, XP_001368145; Gallus gallus, XP_416481; T. guttata, XP_002192984;
D. rerio, NP_001004554; T. nigroviridis, CAF96644; E. coli, NP_415373; M. jann-
aschii, NP_247604.
positions 1 and 2. Also, the proton at position 2 of glutamate
correlates to carbons 4 and 3 of citrate. This spectrum also
allowed us to unequivocally assign the carbon frequencies of
the glutamate moiety. The spectroscopic data are summarized
in supplemental Table S2.
Species Distribution and Evolutionary Tree—Blast searches
indicated that mammalian genomes encode both RIMKLA and
RIMKLB. Only one gene is found in the genomes of the fishes
Tetraodon nigroviridis and Danio rerio and in the two birds
Gallus gallus and Taeniopygia guttata. In birds, it encodes a
product closer to RIMKLB than to RIMKLA. A phylogenetic
tree is shown in Fig. 6. As is indicated by the tree, a gene dupli-
cation has occurred probably after the fish radiation but before
the separation of mammals and birds. However, the precise
order of events could not be determined because of the low
support values for the branching points in this part of the tree.
A striking feature of this tree is that the branches leading to
RIMKLA are longer than those leading to RIMKLB, suggesting
a faster rate of evolution.
␣-carboxylic group of N-acetylaspartate or the ␤-carboxylic
group of citrate. The identity of these two products was estab-
lished by NMR analysis. Synthesized NAAG showed a 1H NMR
spectrum superimposable with that of synthetic ␣-NAAG but
distinctly different from ␤-NAAG. HMBC analysis disclosed
that the bond made by RIMKLB between glutamate and citrate
involved the ␤-carboxylic group of citrate, as in the compound
isolated from tissues (7). The finding that RIMKLB was able to
ligate both NAA and citrate to L-glutamate at similar rates is not
DISCUSSION
Identification of the Reaction Catalyzed by RIMKLA and surprising if one considers that NAA and citrate are almost
RIMKLB—We report in this study the identification of two isosteric, with the ␤-carboxylic group of citrate being in equiv-
enzymes that ligate the ␣-amino group of L-glutamate to the alent position as the ␣-carboxylic group of NAA.
SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
JOURNAL OF BIOLOGICAL CHEMISTRY 29831

Molecular Identification of NAAG and BCG Synthases
Agreement with Previous Results Suggesting That NAAG Is expressed in the placenta of female mice than of male mice (28).
Synthesized by a Ligase—The identification of ligases permit- The significance of this observation is at present unknown.
ting the synthesis of NAAG from NAA and glutamate is con-
Evolution of RIMKLA and RIMKLB—Orthologues of both
sistent with previous observations pointing to the existence of a RIMKLA and RIMKLB are found in all mammals and in Xeno-
ligase catalyzing this reaction from NAA and glutamate rather pus, whereas the fishes and birds genomes comprise only one
than to a ribosome-dependent process. NAAG synthesis from homologous gene, which is closer to RIMKLB than to RIMKLA.
radiolabeled amino acids is insensitive to inhibition of protein These findings suggest that a gene duplication event occurred
synthesis as demonstrated both in spinal sensory ganglia in vivo during or after fish radiation and that the second copy was lost
(11) or in isolated spinal cords (12). Furthermore, labeling stud- in the bird lineage. This is consistent with the finding that the
ies in vivo (24), in spinal cords (12) and in SH-SY5Y human genes that surround the chicken RIMKL gene are homologous
neuroblastoma cells (25) all indicate that NAA is a better pre- to those that surround the mammalian RIMKLB gene, whereas
cursor for NAAG than free aspartate. Finally, almost four the genetic environment of the RIMKLA gene is completely
decades ago Reichelt and Kvamme (19) demonstrated the exist- different (not shown). The ancestral form of RIMKLA and
ence of NAAG synthesis in homogenates of mouse brain and RIMKLB had presumably both activities, consistent with the
showed it to be absolutely dependent on the presence of ATP. presence of both NAAG and ␤-citrylglutamate in fishes (29).
Further attempts to purify this enzyme, have, however, not met The finding that the NAAG/NAA ratio in fish CNS is much
with success. We now have the proof that NAAG is indeed lower than in mammals or amphibians (26, 29) suggests, how-
synthesized by a ligase reaction. This is true also for ␤-citrylglu- ever, that the fish enzyme is not very good at synthesizing
tamate, for which this work represents the first report on its NAAG.
synthesis.
After its separation from RIMKLB, RIMKLA specialized
Agreement between Localization of Products of Reaction and to become a more specific NAAG synthase, consistent with
RIMKLA/B—Because of the reaction they catalyze in vitro, the higher rate of evolution of this enzyme compared with
RIMKLA and RIMKLB are most likely responsible for the syn- RIMKLB. The literature describes other examples of gene du-
thesis of NAAG and ␤-citrylglutamate in vivo. This is in keep- plication events that were followed by a more rapid evolution of
ing with the tissue distribution of these enzymes. RIMKLA is one of the two copies, corresponding to a specialization of the
indeed mostly present in the CNS, particularly in the spinal function of this copy (30, 31). In the present case, this speciali-
cord, in dorsal root ganglions and in the deep nuclei of the zation corresponds to a loss of the ␤-citrylglutamate synthase
cerebellum (Bio-GPS; Allen Atlas). In situ hybridization images activity. This may be advantageous to reach elevated levels of
of mouse brain (Allen Atlas) indicate a marked RIMKLA signal NAAG without diverting too much of an essential metabolic
in motor neurons of the anterior horn and in the dentate intermediate (citrate) or because ␤-citrylglutamate exerts toxic
nucleus of the cerebellum, which is in agreement with the local- effects in some cellular environments or competes with NAAG
ization of NAAG (26). The RIMKLA signal is restricted to a for some essential function.
more limited number of cells than the aspartate N-acetyltran-
NAA and NAAG have been described in the crayfish giant
ferase (NAT8L) signal, consistent with the much wider distri- axon (20), and it is therefore likely that these molecules are
bution of NAA than NAAG in neurons (26). It is likely, there- synthesized in invertebrates by homologues of mammalian
fore, that RIMKLA is expressed only in cells that express NAT8L and RIMKLA/B. Unfortunately, no nuclear genomic
NAT8L. Whether RIMKLA is restricted to neurons or is also sequence is available for the crayfish or any other crustacean.
present in other brain cell types is not known at present. Its high
RIMKLA and RIMKLB as Typical Members of the ATP-Grasp
Km for NAA makes, however, that it can function at a high rate Family—Sequence comparisons indicate that RIMKLA and
only in cells that have a sufficiently high concentration of NAA. RIMKLB both belong to the ATP-grasp superfamily of proteins.
RIMKLB is also present in the CNS, but it seems to be Enzymes of this superfamily catalyze the synthesis of a bond
expressed at the highest level in testis, consistent with the pres- between a carboxylic group and a nucleophile, often an amine,
ence of elevated levels of ␤-citrylglutamate in this organ. As at the expense of ATP, which is converted to ADP and Pi (32).
NAA and NAT8L are absent from this organ, RIMKLB is only Accordingly, RIMKLA was found to form stoichiometric
involved in the synthesis of ␤-citrylglutamate in testis. Interest- amounts of ADP and NAAG. Comparison with E. coli glutathi-
ingly, RIMKLB appears to be up-regulated by 3–7-fold upon one synthase indicates the conservation of several amino acid
differentiation of spermatogonia to leptotene/zygotene sper- residues of the catalytic site. This enzyme has been crystallized
matocytes (27), suggesting that RIMKLB could be involved in with glutathione, ADP, sulfate (which presumably occupies the
spermatogenesis.
position of the ␥-phosphoryl group of ATP) and two Mg2ϩ ions
The presence of both RIMKLB and NAT8L in Purkinje that bind to the phosphates of ADP and the sulfate (33). The
cells in the cerebellum (Allen Atlas) suggests, however, that conserved residues (see Fig. 1) include Gln198 (numbering for
RIMKLB may act to synthesize both NAAG and ␤-citrylgluta- glutathione synthase), whose side chains bind the amino group
mate in some cell types. RIMKLB is most likely responsible for of adenine; Asp208, whose side chain binds O3Ј of the ribose
the synthesis of both compounds in the brain of birds, as there moiety of ADP; Lys160, which binds the ␣-phosphoryl group of
is no RIMKLA in these species. Finally, the presence of ADP; Lys125 and Asn235, which bind the ␤-phosphoryl group;
RIMKLB in placenta (where NAT8L is absent) suggests that the Mg2ϩ binding residues Asp273, Glu281, and Asn283; Arg210
,
␤-citrylglutamate may have a role in this organ. Remarkably, which binds the nucleophilic carboxylic group of glutamyl cys-
the RIMKLB gene has recently been shown to be 3-fold more teine, as well as the sulfate replacing the ␥-phosphoryl group;
29832 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 39•SEPTEMBER 24, 2010

Molecular Identification of NAAG and BCG Synthases
and Arg225, which binds the carboxylic group of glycine. It is
likely that the conserved residues in RIMKLA and RIMKLB
play equivalent roles. These findings indicate that RIMKLA and
RIMKLB presumably have a similar reaction mechanism as glu-
tathione synthase, involving the transient formation of an
acylphosphate intermediate. The structural comparison indi-
cates also that the C-terminal extension of RIMKLA and
RIMKLB are distant from the catalytic domain and have no
equivalent in glutathione synthase. This agrees with our obser-
vation that their removal by proteolytic breakdown does not
affect significantly the kinetic properties of the two ligases.
Their function is presently unknown.
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Conclusion—In conclusion, our work led to the identification
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This should help create cellular models to understand the phys-
iological function of these molecules, and in particular, the
mechanisms by which they are (at least in the case of NAAG)
excreted from cells as well as their physiological role in vivo.
Our work illustrates also the opportunity offered by the avail-
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identify enzymes that, for various reasons, are very difficult to
purify.
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Acknowledgments—We thank Genevie`ve Connerotte and Gae¨tane
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SEPTEMBER 24, 2010•VOLUME 285•NUMBER 39
JOURNAL OF BIOLOGICAL CHEMISTRY 29833

Metabolism:
Molecular Identification of N
-Acetylaspartylglutamate Synthase and β
-Citrylglutamate Synthase
François Collard, Vincent Stroobant, Pedro
Lamosa, Coco N. Kapanda, Didier M.
Lambert, Giulio G. Muccioli, Jacques H.
Poupaert, Fred Opperdoes and Emile Van
Schaftingen
J. Biol. Chem. 2010, 285:29826-29833.
doi: 10.1074/jbc.M110.152629 originally published online July 24, 2010
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