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,Emile Van Schaftingen

doi:10.1074/jbc.m110.152629

Abstract

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.

Highlights

Most in neurons of the anterior horn [1]
Identification of Putative Mammalian NAAG Synthetases—As attempts 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 responding to NAAG were pooled, concentrated to 2 ml in a NAAG is synthesized from N-acetylaspartate and glutamate in lyophilizer, and loaded onto a Bio-Gel P2 column (Bio-Rad; 50 the presence of ATP [12, 19, 20]

Summary

Introduction

Most 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, glutamate carboxypeptidase-III [3], which are anchored to the plasma membrane with their catalytic site oriented toward the outside of the cell. That the effects of NAAG as a neurotransmitter are due to a 0.5% glutamate contamination present in commercial NAAG [5, 6]. ␤-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 decreases with age [7]. NAAG is synthesized in cells even in the presence of protein synthesis inhibitors, from N-acetylaspartate (NAA), suggesting that it is formed by a ligase and not on ribosomes [11, 12]. Our recent success in the identification 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]

Objectives

Methods

Results

Discussion

Conclusion