Publications

• Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione.

  Peracchi A., Veiga-da-Cunha M., Kuhara T., Ellens K.W., Paczia N., Stroobant V., Seliga A.K., Marlaire S., Jaisson S., Bommer G.T., Sun J., Huebner K., Linster C.L., Cooper A.J.L., Van Schaftingen E.

  The mammalian gene <i>Nit1</i> (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known phys ... >> More

  The mammalian gene <i>Nit1</i> (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being ∼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that <i>Nit1</i>-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of <i>Nit1</i>-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in <i>Escherichia coli</i> and other glutathione-producing bacteria. << Less

  Proc. Natl. Acad. Sci. U.S.A. 114:E3233-E3242(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The metabolite repair enzyme Nit1 is a dual-targeted amidase that disposes of damaged glutathione in Arabidopsis.

  Niehaus T.D., Patterson J.A., Alexander D.C., Folz J.S., Pyc M., MacTavish B.S., Bruner S.D., Mullen R.T., Fiehn O., Hanson A.D.

  The tripeptide glutathione (GSH) is implicated in various crucial physiological processes including redox buffering and protection against heavy metal toxicity. GSH is abundant in plants, with reported intracellular concentrations typically in the 1-10 mM range. Various aminotransferases can inadv ... >> More

  The tripeptide glutathione (GSH) is implicated in various crucial physiological processes including redox buffering and protection against heavy metal toxicity. GSH is abundant in plants, with reported intracellular concentrations typically in the 1-10 mM range. Various aminotransferases can inadvertently transaminate the amino group of the γ-glutamyl moiety of GSH to produce deaminated glutathione (dGSH), a metabolite damage product. It was recently reported that an amidase known as Nit1 participates in dGSH breakdown in mammals and yeast. Plants have a hitherto uncharacterized homolog of the Nit1 amidase. We show that recombinant <i>Arabidopsis</i> Nit1 (At4g08790) has high and specific amidase activity towards dGSH. Ablating the <i>Arabidopsis Nit1</i> gene causes a massive accumulation of dGSH and other marked changes to the metabolome. All plant Nit1 sequences examined had predicted plastidial targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. <i>In vitro</i> transcription/translation assays show that both potential translation start codons in <i>Arabidopsis</i> Nit1 were used and confocal microscopy of Nit1-GFP fusions in plant cells confirmed both cytoplasmic and plastidial localization. Furthermore, we show that <i>Arabidopsis</i> enzymes present in leaf extracts convert GSH to dGSH at a rate of 2.8 pmol min^-1 mg^-1 in the presence of glyoxalate as an amino acceptor. Our data demonstrate that plants have a dGSH repair system that is directed to at least two cellular compartments via the use of alternative translation start sites. << Less

  Biochem. J. 476:683-697(2019) [PubMed] [EuropePMC]