title: Molecular identification of N-acetylaspartylglutamate synthase and beta-citrylglutamate synthase.
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.
authors.affiliation: Laboratory of Physiological Chemistry, de Duve Institute and Université Catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: François
authors.initials: F
authors.lastname: Collard
authors.forename: Vincent
authors.initials: V
authors.lastname: Stroobant
authors.forename: Pedro
authors.initials: P
authors.lastname: Lamosa
authors.forename: Coco N
authors.initials: CN
authors.lastname: Kapanda
authors.forename: Didier M
authors.initials: DM
authors.lastname: Lambert
authors.forename: Giulio G
authors.initials: GG
authors.lastname: Muccioli
authors.forename: Jacques H
authors.initials: JH
authors.lastname: Poupaert
authors.forename: Fred
authors.initials: F
authors.lastname: Opperdoes
authors.forename: Emile
authors.initials: E
authors.lastname: Van Schaftingen
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry / Q000502:physiology; D002460:Cell Line; D004151:Dipeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D051379:Mice; D046908:Peptide Biosynthesis, Nucleic Acid-Independent / Q000502:physiology*; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D004151:Dipeptides; D012269:Ribosomal Proteins; C027172:isospaglumic acid; D010453:Peptide Synthases