The mammalian nitrilase (Nit) protein is a member of the nitrilase superfamily whose function remains to be characterized. We now show that the nitrilase family member 2 gene (NIT2) is ubiquitously expressed in multiple tissues and encodes protein mainly distributed in the cytosol. Ectopic expression of Nit2 in HeLa cells was found to inhibit cell growth through G2 arrest rather than by apoptosis. Consistent with this, proteomic and RT‐PCR analyses showed that Nit2 up‐regulated the protein and mRNA levels of 14‐3‐3σ, an inhibitor of both G2/M progression and protein kinase B (Akt)‐activated cell growth, and down‐regulated 14‐3‐3β, a potential oncogenic protein. Genotype analysis in four types of primary tumor tissues showed 12.5–38.5% allelic imbalance surrounding the NIT2 locus. The results demonstrated that NIT2 plays an important role in cell growth inhibition and links to human malignancies, suggesting that Nit2 may be a potential tumor suppressor candidate.

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

Background: Human Nit2/-amidase is a putative tumor suppressor. Results: Both the catalytic triad and loop 116 -128 of hNit2 play an essential role in the enzyme-substrate binding and enzymatic activity. Conclusion:The results of MD simulations are consistent with the kinetic analysis obtained with substrates ␣-ketoglutaramate and succinamate. Significance: This work provides the basis for new areas of research into tumor glutamine metabolism and hyperammonemic diseases.

Sialidases catalyse the removal of terminal sialic acids from a range of glycoproteins, glycolipids and oligosaccharides. They have been found in bacteria, viruses and parasites, where they play important roles in pathogenesis and/or microbial nutrition, and in mammalian cells, where they modulate cell-surface glycosylation associated with a range of cellular activities. Clostridium perfringens, a causative agent of gas gangrene and peritonitis in humans, possesses three sialidases: nanH, nanI and nanJ, with molecular weights of 42, 77 and 129 kDa, respectively. The two larger enzymes are secreted by the bacterium and are involved in the pathogenesis and nutrition of Clostridium. As part of a study to examine the structures of all three enzymes, crystallization of the 77 kDa nanI isoenzyme was attempted. The expressed full-length protein was found to degrade easily; a stable 50 kDa catalytic domain was therefore subcloned. This domain was overexpressed in Escherichia coli and produced crystals belonging to space group P2 1 2 1 2 1 , with unit-cell parameters a = 96.98, b = 69.41, c = 72.69 A Ê and one monomer per asymmetric unit. The crystals diffract to at least 0.92 A Ê . A molecular-replacement solution was obtained using the catalytic domain of the sialidase from the leech Macrobdella decora.

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