Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism

Abstract

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms.

Two novel fish paralogs provide insights into the Rid family of imine deaminases active in pre-empting enamine/imine metabolic damage

“…5, upper panel). This behavior has been previously reported also for Ch RidA 25 , indicating that both goat RidA and salmon RidA-1 have a remarkable resistance to thermal unfolding. Ss RidA-1 thermal stability was further investigated by repeating the experiment in the presence of increasing concentrations of urea (Fig.…”

“…5, lower panel). The calculated value of 100.2 °C is slightly lower than that calculated for Ch RidA (104 °C) 25 .…”

“…The activity of RidA enzymes was tested using FAD-dependent L-amino acid oxidases that can generate 2-imino acids www.nature.com/scientificreports www.nature.com/scientificreports/ (IA) upon oxidation of the corresponding L-amino acids 8,9,25 . The activity of Ss RidA-1 and Ss RidA-2 on a broad spectrum of IA was compared to that of goat RidA ( Ch RidA), used as a reference being the best-characterized animal RidA 25 . Both Ss RidA-1 and Ss RidA-2 were active as imine deaminases but striking differences in substrate specificity between the isozymes were observed as highlighted in Fig.…”

“…In the frame of a broader study on eukaryotic RidA, we previously characterized the biochemical properties of goat UK114 ( Ch RidA), an acid-soluble homotrimeric liver protein that became the object of intense studies more than twenty years ago due to its peculiar immunogenic properties, well before the proof of its biochemical activity was obtained 25 . Here, we identified Ch RidA homologs in teleosts (bony fish), a class of vertebrates in which, to the best of our knowledge, the Rid proteins have never been studied.…”

Integrated Metabolomics and Transcriptomics Suggest the Global Metabolic Response to 2-Aminoacrylate Stress in Salmonella enterica

“…The RidA paradigm of enamine stress in Salmonella enterica defines a metabolic model with clearly defined local effects that result in predictable global consequences. Members of the broadly conserved RidA protein family are responsible for the hydrolysis of enamine/imine species, which are generated as intermediates in a variety of biochemical reactions [15][16][17][18][19][20] (Figure 1). In the absence of RidA, the enamine 2-aminoacrylate (2AA), a well-characterized inhibitor of multiple pyridoxal 5′-phosphate (PLP)-dependent enzymes, accumulates ( Figure 1).…”