Background It really is now recognized that enzymatic or chemical substance side-reactions may convert regular metabolites to useless or toxic ones and a collection of enzymes exists to mitigate such metabolite harm. activity. Analysis from the chromosomal framework of bacterial RidA genes exposed clustering with genes for threonine dehydratase and additional pyridoxal 5′-phosphate-dependent enzymes, which suits using the known RidA imine hydrolase activity. Clustering was also apparent between Rid family members genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate rate of metabolism. Biochemical assays demonstrated that RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate. Conclusions Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by 1-Azakenpaullone amine oxidases as well as by pyridoxal 5′-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. 1-Azakenpaullone Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users. molecular chaperone activity for DUK114 , endoribonuclease activity for rat L-PSP , and specialist chorismatase  and 2-aminomuconate deaminase [17,18] activities in bacterial aromatic metabolism. There is good reason to think that YjgF/YER057c/UK114 family proteins have other C and widespread C roles in metabolic processes. The family takes place atlanta divorce attorneys organism in every domains of lifestyle almost, and some species encode multiple members. has eleven. Although YjgF/YER057c/UK114 proteins or domains are uniformly small (~130 amino acids), their sequences are diverse and some members share <8% sequence identity [19,20]. The phylogenetic distribution pattern and sequence diversity strongly imply multiple functions, and several have been suggested in pyrimidine degradation [21,22], mitochondrial maintenance [23,24], and metabolic regulation . To explore additional functional roles, we made phylogenetic and comparative genomic analyses of the YjgF/YER057c/UK114 family. This work enabled prediction of two novel functions for RidA proteins, for which experimental support was obtained by biochemical, genetic, and metabolomic approaches. Results Phylogenetic analysis and nomenclature of the YjgF/YER057c/UK114 family YjgF/YER057c/UK114 family proteins are split into eight subfamilies in the NCBI Conserved Domain name Database (see cd00448: YjgF_YER057c_UK114_family), which uses a position-specific scoring matrix to determine conserved domain name footprints that imply potential functional sites . Each subfamily could accordingly have distinct functional activities. We henceforth refer to the whole family as the Rid family, to the subfamily made up of the characterized RidA proteins 1-Azakenpaullone as the RidA subfamily, and to the other seven subfamilies as Rid1 through Rid7 (Table?1). This nomenclature meshes with that used in previous studies [8-11]. A subset of RidA proteins (which we term 3x-RidA) has three RidA domains fused in tandem (e.g. EF_0115 in V583). As Rid proteins typically assemble into trimers  (Physique?1A) the 3x-RidA proteins are presumably covalently linked trimer models. Other arrangements such as fusions of two or four Rid domains occur, but are rare and so were excluded from this study. Table 1 Rid subfamily nomenclature Physique 1 Sequence features of Rid family members. (A) Common trimeric organization of a RidA protein, TdcF. (B) TdcF active site with bound serine molecule. Residues of adjacent monomers are colored reddish or blue. (C) Sequence logos show the conservation ... Crystal structures and mutagenesis studies have recognized functionally important residues in the RidA subfamily. The highly conserved Arg107 forms salt bridges with the carboxylate oxygens of benzoate in 1-Azakenpaullone human hp14.5  and the corresponding Arg105 hydrogen bonds with the carboxylate oxygens of serine, threonine, and 2-oxobutanoate in Rabbit polyclonal to GHSR TdcF (paralogous to RidA)  (Determine?1B). Changing this arginine to alanine almost completely abolished imine-hydrolyzing activity in herb and RidA proteins [8,9]. It has been suggested that conserved Tyr17 and Glu120 residues of TdcF also play a role in substrate binding and positioning of a water molecule utilized for imine hydrolysis, but replacing the corresponding residues with alanine experienced little effect on 1-Azakenpaullone the activity of RidA . We analyzed residue conservation for all the Rid family proteins outlined in the NCBI Conserved Domain name Database, spending particular attention to the predicted active site residues (Physique?1C). The Rid1, Rid2, and Rid3 subfamilies retain the conserved arginine and glutamate residues found in RidA. The Rid4, Rid5, Rid6, and Rid7 subfamilies have the glutamate residue however the arginine residue is certainly changed by tryptophan in Rid4 and Rid7, and it is variable in Rid6 and Rid5. The tyrosine is certainly conserved, or conservatively.