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highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group).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.metabolite repair | deaminated glutathione | amidase | aminotransferases

Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight

“…That the growth effects of ablating pxpABC in B. subtilis, and also in E. coli (supplemental Fig. S2), are modest conforms to a pattern noted for other microbial damage-control genes (27,(35)(36)(37).…”

Escherichia coli as a host for metabolic engineering

“…The promiscuous nature of many transaminases produces a deaminated glutathione molecule which adversely affects function of glutathione-dependent enzymes. A eukaryotic protein, Nit1, was recently identified that can hydrolyze deaminated glutathione into cysteinylglycine and α-ketoglutarate (Peracchi et al, 2017). In other cases, 5,10-Methenyltetrahydrofolate can form a 5-Formyltetrahydrofolate side product from serine hydroxymethyltransferase (Stover and Schirchs, 1990), and glutamine or glutamate can spontaneously cyclize to form 5-oxoproline (Park et al, 2001).…”

Enzyme promiscuity, metabolite damage, and metabolite damage control systems of the tricarboxylic acid cycle

“…For example, inactivation of 6‐NAD(P)H oxidase results in the accumulation of 4,5,6‐NAD(P)H 3 , which is itself a damage product of 6‐NAD(P)H (i.e., the metabolite damage product of a damaged metabolite) [115]. Even if a damaged metabolite is detected, elucidating the structure can be challenging [11,122]. It is also difficult to predict which promiscuous activities or spontaneous reactions are likely to occur in cells [12,123], and more difficult still to predict which of the potential metabolite damage products are deleterious, and thus, likely to be enzymatically repaired.…”