1. J Biol Chem. 2010 Sep 24;285(39):29826-33. Epub 2010 Jul 24.

Molecular identification of N-acetylaspartylglutamate synthase and
beta-citrylglutamate synthase.

Collard F, Stroobant V, Lamosa P, Kapanda CN, Lambert DM, Muccioli GG, Poupaert
JH, Opperdoes F, Van Schaftingen E.

Laboratory of Physiological Chemistry, de Duve Institute and Université
Catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium.

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.


PMCID: PMC2943312 [Available on 2011/9/24]
PMID: 20657015 [PubMed - indexed for MEDLINE]