Members of the YjgF/YER057c/UK114 Family of Proteins Inhibit Phosphoribosylamine Synthesis in Vitro

Abstract

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

Conserved YjgF Protein Family Deaminates Reactive Enamine/Imine Intermediates of Pyridoxal 5′-Phosphate (PLP)-dependent Enzyme Reactions

“…Diversity among YjgF Homologs-YjgF homologs from five diverse organisms were found to have the same activity as the S. enterica protein when measured in assays described here and previously (13). The diversity of homologs that have activity in the assays minimizes the possibility that a protein-protein interaction is required for YjgF activity.…”

“…This activity has two anticipated consequences in vivo: 1) increasing the hydration rates in low water environment to improve product generation and 2) removing reactive metabolites. We suggest that the diverse phenotypes described for ridA (yjgF) mutants in S. enterica (10,11,13,33) result from the accumulation of enamine/imine intermediates and that the nucleophilic nature of these metabolites allows them to react with and modify cellular targets, altering their function. The plausibility of this scenario is supported by literature reports that the aminoacrylate enamine derived from serine can inactivate a number of enzymes in vitro (43,44).…”

“…Past work, primarily in S. enterica, suggested that the diverse phenotypes of yjgF mutants reflected a global metabolic role for this protein family (11)(12)(13)33). The results reported herein define an enamine/ imine deaminase activity associated with the YjgF protein and homologs from all domains of life, including humans.…”

“…Enzyme Purifications-IlvA and YjgF were purified using C-terminal His 6 tags and nickel ion affinity chromatography, as described previously (13). Variants YjgF Y17F , YjgF R105A , YjgF E120K , and YjgF E120A and protein homologs from B. subtilis (YabJ) and P. furiosus (PF0668) were purified following the same protocol as for YjgF.…”

Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases

“…We also analyzed the imine deaminase activity for the mutant of Der f 34, whose conserved seven amino acid residues for enamine/imine deaminase activity were substituted for alanine. RidA protein from Salmonella enterica (NCBI GenBank TM accession number AF095578) and a nonfunctional variant (RidA R105A ) were used as a positive and negative control for the imine deaminase assay (27,36). The imine deaminase activity was determined as an inhibitory activity to produce semicarbazone, which was produced by a reaction with imine and semicarbazide (37).…”