NAD(P)HX repair deficiency causes central metabolic perturbations in yeast and human cells

Abstract

reading frame; PSAT1, phosphoserine transaminase 1. Keywords-metabolite repair, NAD(P)HX dehydratase, NAD(P)HX epimerase, Saccharomyces cerevisiae, inborn errors of metabolism.Conflict of interest-The authors declare that they have no conflicts of interest with the contents of this article.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/302257 doi: bioRxiv preprint first posted online Apr. 16, 2018; 2 ABSTRACT NADHX and NADPHX are hydrated and redox inactive forms of the NADH and NADPH cofactors, known to inhibit several dehydrogenases in vitro. A metabolite repair system that is conserved in all domains of life and that comprises the two enzymes NAD(P)HX dehydratase and NAD(P)HX epimerase, allows reconversion of both the S-and R-epimers of NADHX and NADPHX to the normal cofactors. An inherited deficiency in this system has recently been shown to cause severe neurometabolic disease in children. Although evidence for the presence of NAD(P)HX has been obtained in plant and human cells, little is known about the mechanism of formation of these derivatives in vivo and their potential effects on cell metabolism. Here, we show that NAD(P)HX dehydratase deficiency in yeast leads to an important, temperature-dependent NADHX accumulation in quiescent cells with a concomitant depletion of intracellular NAD + and serine pools. We demonstrate that NADHX potently inhibits the first step of the serine synthesis pathway in yeast. Human cells deficient in the NAD(P)HX dehydratase also accumulated NADHX and showed decreased viability. In addition, those cells consumed more glucose and produced more lactate, potentially indicating impaired mitochondrial function. Our results provide first insights into how NADHX accumulation affects cellular functions and pave the way for a better understanding of the mechanism(s) underlying the rapid and severe neurodegeneration leading to early death in NADHX repair deficient children.

NAD(P)HX dehydratase (NAXD) deficiency: a novel neurodegenerative disorder exacerbated by febrile illnesses

“…More specifically, it can be predicted that the brain, which has a very high and constant demand for energy supply generated by mitochondria, would be particularly vulnerable to impaired NAD(P)HX repair and as such, the NAXE and NAXD enzymes would be critical to support normal brain function. In apparent contradiction with those predictions, however, knocking out NAD(P)HX repair enzymes in other organisms such as yeast, bacteria and plants, has so far only revealed subtle growth phenotypes, if any, at least under standard growth conditions (Breslow et al, 2008;Hillenmeyer et al, 2008;Nichols et al, 2011;Colinas et al, 2014;Niehaus et al, 2014;Becker-Kettern et al, 2018).…”

“…Missense variants were introduced into wild-type cNAXD and proteins expressed and purified from a bacterial overexpression system as detailed in the Supplementary material. The S-NADHX substrate was synthesized and purified as previously described (Becker-Kettern et al, 2018) and NAXD enzyme kinetic properties determined using a spectrophotometric assay (Marbaix et al, 2011). Protein thermostability was tested by pre-incubation of purified protein at different temperatures (30-47 C).…”

“…The damaged cofactors cannot act as electron carriers and have been shown in vitro to inhibit several key dehydrogenase enzymes (Yoshida and Dave, 1975;Prabhakar et al, 1998). In Saccharomyces cerevisiae, in vitro and in vivo evidence for an inhibitory effect of NADHX on 3-phosphoglycerate oxidoreductase, catalysing the initial step of the serine biosynthesis pathway, has been obtained recently (Becker-Kettern et al, 2018). Therefore, NAD(P)HX can be expected to be toxic to cells, and detoxification by a metabolite repair system is critical.…”

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

“…Breaches of specificity in metabolic enzymes, and in particular the central ones, can be highly deleterious. The identification of repair enzymes that catabolize the resulting damage metabolites provides some indications about the nature of the potential competing substrates . However, in the majority of cases, the potential threat substrates remain unknown.…”

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“…El mismo sistema de reparación actúa sobre las formas modificadas de las dos coenzimas y se ha identificado en todos los dominios de la vida. Su deficiencia causa problemas neurodegenerativos letales en mamíferos (Van Bergen et al 2019, Becker-Kettern et al 2018, depleción de la reserva de serina en levaduras (Becker-Kettern et al 2018), o dificultad en la adaptación a estrés en Bacillus subtilis (Petrovova et al 2014). Curiosamente la carencia de la deshidratasa no parece causar problemas en plantas (Colinas et al 2014).…”