## 3 Discussion  
In this work we defined that UK114 is a deiminase that exhibits properties that attribute it to the RidA subfamily, as also suggested by the presence of the conserved Arg107 (equivalent of Arg105 of other RidA) in the signature consensus pattern (Figure 2 and [6]). The specificity of UK114 toward various \(\alpha\)-imino acids was compared to that reported for other RidA proteins. In the following, the amino acid is indicated instead of the imino acid for simplicity. The specificity study revealed a preference in the following order: Ala > Leu = L-Met > L-Gln. Little or no activity was detected using L-Phe and L-His. The data on L-Leu, L-Met, L-Gln, L-Phe, and L-His are similar to those that are reported for the RidA of _S. enterica_[6; 15]. In other organisms, Rid2 and Rid3 proteins are active on L-Phe and L-His, but no homologs of these Rid subfamilies were detected in eukaryotes [15]. Interestingly, by generating the imino acids with L- and D-amino acid oxidases and the corresponding L- and D-amino acid under identical conditions, we showed--for the first time--that the highest specificity is indeed observed with 2-iminopyruvate (2-iminopropionate). This compound is the proposed physiological substrate of RidA proteins being the tautomer of the toxic 2-aminoacrylate generated by serine/threonine dehydratase and cysteine desulfhydrase [2; 3; 5]. It should be noted that--so far--the latter enzymes have been used to generate the RidA substrate, leaving some uncertainty about the nature of its actual substrate: the enamine or the imino acid. Therefore, the finding of activity with the product of the reaction of LAAO and DAAO and L- and D-Ala (or L- and D-Leu), respectively, clarifies that the actual RidA substrate is most likely the imino acid, rather than the corresponding enamine. In addition, our results provide a simple assay for testing the deiminase activity of Rid proteins, even with the proposed physiological substrate (2-iminopyruvate) with LAAO and DAAO that are commercially available. The RidA protein UK114 is likely to have provided a solution to the phenomenon called metabolite damage, which is caused by accumulation of reactive enamines/imines, also in mammalian cells. This basic function appeared in early evolution (before the divergence of the three domains of life) and was maintained, an index of its important biological role. Since also PLP-dependent enzymes appeared early in evolution [48; 49], it can be hypothesized that the selective pressure on RidA proteins was imposed by the co-presence of reactions catalyzed by PLP-dependent enzymes that generated toxic compounds, such as 2 AA, and others that are sensitive to 2-AA [6]. The accumulation of 2 AA was reported in cells devoid of RidA activity and 2 AA stress is now recognized as an unbalanced metabolic condition that can cause several phenotypic defects. Thus, RidA proteins play an important function by contributing to the metabolic homeostasis on which cells rely on to survive. The fact that UK114 is maximally expressed in the liver, which is a crucial organ for amino acid metabolism, could be further explored by using (e.g.,) knock-out mice to understand the biological role of RidA proteins in mammals. Whether UK114 serves to hydrolyze imines produced by enzymes other than serine/threonine dehydratase or cysteine desulfhydrase, such as for example LAAO and DAAO, monoamine oxidases, or other amine oxidases, will need to be established to further clarify both its mechanistic features and its biological role in various organisms and tissues. The latter would be very interesting in the light of the known or proposed roles of these enzymes in fundamental processes and disease [50; 51; 52; 53]. However, the feasibility of these studies will depend on the ability to set up robust activity assays, which will also allow for carrying out in depth mechanistic work.