Identification of the putative tumor suppressor Nit2 as ω-amidase, an enzyme metabolically linked to glutamine and asparagine transamination

Abstract

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is ω-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated ω-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are α-ketoglutaramate and α-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of α-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating α-keto-γ-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess α-ketoglutaramate may be neurotoxic. Moreover, α-ketosuccinamate is unstable and is readily converted to a number of hetero aromatic compounds that may be toxic. Thus, an important role of ω-amidase is to remove potentially toxic intermediates by converting α-ketoglutaramate and α-ketosuccinamate to biologically useful α-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of ω-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor

Structural Insights into the Catalytic Active Site and Activity of Human Nit2/ω-Amidase

“…Protein overexpression was induced with 2 mM isopropyl 1-thio-␤-D-galactopyranoside and maintained for 6 h; the overexpressed protein in the crude protein fraction obtained from the periplasmic fraction was further purified. We chose KGM, SM, and 3-PPN for assay of the enzymatic activity of hNit2/-amidase (11,23,31). The proteins were then subjected to kinetic analysis.…”

“…However, mammals also possess another pathway (the glutaminase II pathway) for the metabolism of glutamine. In the glutaminase II pathway, glutamine is transaminated to ␣-ketoglutaramate(KGM), 3 followedby-amidase-catalyzedhydrolysis of KGM to ␣-ketoglutarate (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Similarly, transamination of asparagine yields ␣-ketosuccinamate (KSM), followed by -amidase-catalyzed hydrolysis of KSM to oxaloacetate (1)(2)(3).…”

“…In the methionine salvage pathway, ␣-keto-␥-methiolbutyrate is formed from 5Ј-methylthioribose. ␣-keto-␥-methiolbutyrate is then converted to methionine by the glutaminase II pathway, thereby linking sulfur metabolism to the tricarboxylic acid cycle (11). KSM is unstable and can give rise to a plethora of aromatic heterocyclic compounds.…”

“…A link between cancer biology and the glutaminase II pathway has recently been strengthened by the finding that nitrilase-like protein 2 (Nit2), which was previously shown to be a putative tumor suppressor (22), is identical to -amidase (10,11,23). On the basis of sequence analysis, Pace et al reported that Nit2 belongs to the nitrilase (Nit) superfamily (24,25).…”

Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

“…Knowing the function of these putative enzymes, particularly if they are highly conserved, may reveal important aspects of intermediary metabolism that have remained unexplored until now. The purpose of the present work was to determine the catalytic role of nitrilase-like protein 1 (Nit1), a highly conserved protein with still unknown function (1-6) but homologous to Nit2 (ω-amidase) (5,6), an enzyme whose activity is closely linked to transamination reactions.…”