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Nitl is a protein that belongs to the family of Nitrilases that still has a mysterious physiological function. However, Nitl has a close homologue whose function is well known, the ro-amidase Nit2 with which it shares 37% identity and that hydrolyzes a-ketoglutarate in a-ketoglutaramate and ammonia. lt is therefore likely that Nit l acts on a substrate that is structurally similar to that of Nit2. However, there is no other a-ketoglutaryl compound that is a well-known intermediate in the metabolic pathways that are described. This observation led us to consider the question of the physiological function of Nit l assuming it could be an enzyme involved in the metabolism of "abnormal metabolites". Nit l could for example "repair" errors made by enzymes that catalyse reactions using substrates that are structurally related to a-ketoglutarate. A first potential candidate for one of these enzymes is a mitochondrial protein that activates glutarate or succinate to glutarylCoA or succinylCoA, and which could also use the a-ketoglutarate, abundant in the rnitochondria, to form the a-ketoglutarylCoA. Nit l could then hydrolyse this compound, preventing its accumulation on the one hand and, on the other hand allowing the recovery of a-ketoglutarate and CoASH. The CoA transferase that catalyzes this reaction has never been molecularly identified, but there is a metabolic deficiency, the glutaric aciduria type III, that is characterised by an accumulation of glutaric acid in urine, that was linked to homozygous mutations in the gene C70RFJ O. It is therefore likely that C70RFJ O encodes the succinylCoA: glutarate CoA transferase. We expressed the recombinant C70RF10 in Escherichia coli, purified it and showed that, indeed, it activates glutarate or succinate to glutarylCoA or succinylCoA, respectively. We also studied the effect of the Arg336Trp replacement that was described in individuals with glutaric aciduria type III, by showing that it drastically reduced the production of an active recombinant enzyme. Subsequently, we analysed in detail the specificity of the transfer of CoASH and showed that C70RF l 0 can use many dicarboxylic acids as CoASH acceptors, and in particular that a­ ketoglutarate was also activated to a-ketoglutarylCoA.This allowed us to show that a-ketoglutarylCoA was indeed a substrate of Nit l, and that among all dicarboxylylCoAs that are made by C70RF 10, it is the only one that is hydrolyzed by Nit l. However, a-ketoglutarylCoA is not only a substrate for Nit l but also for Nit2, despite a rate of hydrolysis, which is 1.5 times, lower than for Nit l. This observation indicates that a-ketoglutarylCoA is not the physiological substrate of Nitl, but shows undeniably that the presence of a ketone derivative on the a-carbon of the dicarboxylyls compounds that we tested is essential for substrate recognition.Thus, we also explored the possibility that glutamate cysteine ligase, the first enzyme of the glutathione synthesis pathway, which catalyzes the condensation reaction between the y-carbon of glutamate and a­ amine of cysteine, may catalyse by mistake a similar reaction using a-ketoglutarate. ln this case, the a-ketoglutarylcysteine formed could be a substrate for Nit l. However, our experiments showed that a-ketoglutarylcysteine was neither hydrolysed by Nit l nor by Nit2. The detailed analysis of the structures of the catalytic sites of Nit l and Nit2 that we made shows that they are similar, but that Nit l could accommodate a larger substrate molecule than Nit2. Furthermore, our experiments have shown that the subcellular localization of Nit l and Nit2 are similar and appear to be both in the cytoplasm and in the mitochondria. The hypothesis that follows and that should be tested is that Nitl intervenes in the degradation of cyclic a-ketoglutaramate, which is the most abundant form, white Nit2 hydrolyses the linear molecule. This could be done by means of an activation of the cycle via the transfer of a nucleotydyl group by a yet unknown enzyme.The derivative thus formed could then be the potential substrate of Nitl.

nitrilase --- Glutaryl-CoA Dehydrogenase