PurF-Independent Phosphoribosyl Amine Formation inyjgFMutants ofSalmonella entericaUtilizes the Tryptophan Biosynthetic Enzyme Complex Anthranilate Synthase-Phosphoribosyltransferase

Abstract

In Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine (PRA) is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). A null mutation in yjgF allows PurF-independent PRA formation by an unknown mechanism. The tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase, composed of the TrpD and TrpE proteins, was shown to be essential for PRA formation in strains lacking both yjgF and purF. The activity generating PRA in a yjgF mutant background has features that distinguish it from the TrpDE-mediated PRA formation shown previously for this enzyme in strains with an active copy of yjgF. The data presented here are consistent with a model in which the absence of YjgF uncovers a new catalytic activity of TrpDE.

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

“…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.…”

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

“…Preliminary results suggest the X-NH 3 metabolite is unstable, perhaps favoring an explanation involving a reaction intermediate rather than a byproduct. 5 The ability of YjgF protein to inhibit the TrpD-dependent PRA formation in vitro was predicted by both in vivo phenotypes and the working model for this class of proteins (8,10,11). The inhibition of PRA formation by YjgF displayed several features that provided insights into the function of this protein.…”

“…The strain carrying trpE8 was provided by J. R. Roth. Deletion of the trp operon was generated by linear recombination (18) and has been described (8). Plasmid pTrpD (pIR-DW) contained wild-type trpD in pSU19 under the control of the plasmid encoded lac promoter and has been described (19).…”

“…A strain lacking purF, in combination with a null mutation in gnd, encoding gluconate-6-phosphate dehydrogenase, is unable to generate PRA and thus cannot grow in the absence of thiamine (6,7). However, if a third gene, yjgF, is eliminated, PRA synthesis and thus thiamine-independent growth are restored (2,8).…”

“…Past work implicated the tryptophan biosynthetic complex anthranilate synthase-phophoribosyltransferase (EC 4.1.3.27, 2.4.2.18) in PurF-independent PRA formation in a yjgF background (8). During tryptophan biosynthesis, the TrpDE complex catalyzes the conversion of chorismate to anthranilate while deaminating glutamine, and subsequently TrpD combines anthranilate and phosphoribosyl pyrophosphate (PRPP) to form phosphoribosyl anthranilate and inorganic phosphate (12,13).…”