PurF-independent phosphoribosyl amine formation in yjgF mutants of Salmonella enterica utilizes 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.

FIG. 1.

Thiamine pyrophosphate biosynthetic pathway in S. enterica. (A) Simplification of the biosynthetic pathway for thiamine production. Emphasis is placed on the two reactions relevant to this work. The reactions are catalyzed by the gene product indicated. The PurD reaction has been used in the coupled assay to monitor PRA formation in vitro (36). (B) Reaction catalyzed by the TrpDE enzyme complex that results in an intermediate in the biosynthesis of tryptophan.

FIG. 2.

The tryptophan operon in S. enterica, illustrating the three constructed deletions. A series of three deletions (designated by allele numbers) of various portions of the tryptophan operon were introduced into a purF gnd yjgF mutant background (strain DM7436), and the resulting growth phenotypes on media containing gluconate, adenine, and tryptophan in the presence and absence of exogenous thiamine are presented.

FIG. 3.

TrpDE-dependent PRA formation in a yjgF mutant is not sensitive to tryptophan. Growth at 37°C in glucose adenine tryptophan medium with arabinose (1 mM) added was monitored (A₆₅₀ versus time). Two parental strains and three plasmids are involved. (A) Data for strains with a wild-type allele of yjgF, including strains DM9227 [purF gnd (pBAD)] ([circo]), DM9228 [purF gnd (pBAD-TrpED)] (▴), and DM9229 [purF gnd (pBAD-TrpE(P289T)D)] (▪). (B) Data for strains lacking yjgF, including strains DM9230 [purF gnd yjgF (pBAD)] (○), DM9231 [purF gnd yjgF (pBAD-TrpED)] (▴), and DM9232 [purF gnd yjgF (pBAD-TrpE(P289T)D)] (▪).

FIG. 4.

The trpE3613 allele is detrimental to thiamine synthesis in a yjgF mutant. Growth of DM7436 (purF gnd yjgF) at 37°C in glucose adenine, glucose adenine tryptophan, and glucose adenine tryptophan thiamine was compared to that of DM7435 (purF gnd yjgF trpE3613) under similar growth conditions (A₆₅₀ versus time).

FIG. 5.

Working model for the influence of a yjgF mutation on purF-independent PRA synthesis. The features of this model are described in the text (see Discussion). Depicted is a generic enzyme in central metabolism that has a 2^o reaction generating metabolite X. The two distinct TrpDE-dependent PRA-forming reactions supported by the data herein are depicted and labeled 1 and 2. Activity 1 is shown to generate PRA from substrate X that is normally sequestered and/or eliminated by YjgF. Activity 2 has been described previously (36) and is considered to be a weak activity that can be enhanced by overexpression or mutation.