Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione

Abstract

The mammalian gene Nit1 (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highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group).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 Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-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 Escherichia coli and other glutathione-producing bacteria.

Keywords: amidase; aminotransferases; deaminated glutathione; metabolite repair.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

The Nit2 reaction and the working hypotheses tested in this work about Nit1 activity. (A) The known coupled reactions catalyzed by glutamine transaminases and ω-amidase (Nit2). The transamination of glutamine yields α-KGM, the linear form of which equilibrates with a cyclic form (two potential anomers). Nit2 catalyzes the hydrolysis of the open form to α-KG and ammonia. (B) An analogous process, in which the transamination of GSH to dGSH is followed by hydrolysis of dGSH to α-KG and cysteinylglycine. (C) α-KG–CoA, formed by SUGCT (C7orf10), is another potential substrate for Nit1.

Fig. 2.

Accumulation of dGSH in Nit1-deficient HAP1 cells and in NIT2-deficient yeast. (A) HAP1 cell extracts were prepared from two cell lines deficient in Nit1, from one HAP1 cell line deficient in Nit2 and from three control cell lines. Among these, two were cell lines that had been submitted to the CrispR/Cas9 gene inactivation procedure, but yielding no modification of the Nit1 gene; this was done to exclude the possibility of any off-target effect of the guide RNAs used that may lead to dGSH accumulation. (B) WT S. cerevisiae and mutants lacking either scNit1 (NIT2-KO) (Table 1) or scNit2 (NIT3-KO) were cultivated in minimal defined medium with 1% glucose. Samples were collected after 15 h from starting the culture, corresponding to the late exponential growth phase. Intracellular metabolites were extracted, analyzed by LC/MS and dGSH and GSH were quantitated as above. Intracellular concentrations were calculated as detailed in Materials and Methods. Means ± SEM of three biological replicates are shown. A modest but statistically significant (P < 0.05) increase in the intracellular dGSH concentration was reproducibly observed in NIT3-KO cells compared with WT. The NIT3-KO cells appeared to accumulate substantial amounts of α-KGM (≥300 μM), and it is possible that the observed increase in dGSH is secondary to this accumulation.

Fig. 3.

LC/MS detection of dGSH in the urine of Nit1-deficient mice. LC/MS chromatograms showing the presence of dGSH in the urine (500-fold diluted) of a Nit1-KO mouse (red line) and a WT mouse (green). The blue chromatogram refers to urine from a WT mouse spiked with 25 μM purified dGSH. (Inset) MS2 spectrum of dGSH found in the Nit1-KO urine sample, mirrored with the MS2 spectrum generated from the standard.

Fig. 4.

GC/MS detection of a dGSH derivative in the urine of Nit1-deficient mice. Urine samples were treated with the TMS reagent BSFTA/TMCS [N,O-bis(trimethylsilyl) trifluoroacetamide-trimethylchlorosilane] (SI Appendix, Supplementary Materials and Methods). (A) GC/MS total ion current chromatogram showing the presence of a major peak (with retention time 11.97 min and m/z 398 for the apparent molecular ion) in the urine of a Nit1-KO mouse. The same peak was observed in all of the urine samples from Nit1-KO mice. (B) In contrast, the peak was undetectable in the urine of control mice. (C) GC/MS analysis of purified dGSH shows a peak with the same retention time and mass spectrum (see also SI Appendix, Fig. S5), indicating that this is a derivative of dGSH. (D) Mass spectrum of the compound present in the urine of Nit1-KO mice. The molecular ion shifted to m/z 416 upon silylation with N,O-Bis (trimethyl-d9-silyl)acetamide (d9-BSA) (SI Appendix, Fig. S5C), indicating that the ion is M+ and contains two TMS moieties. High-resolution MS indicated the elemental formula C16H26N2O6Si2 (exact mass 398.13892; calculated mass 398.13294; millimass error 5.98 and unsaturation of 7.0), implying for the underivatized compound MW = 254 and the elemental formula C10 H10 N2 O6. The Inset shows the proposed structure for the dGSH derivative.