Anthranilate Synthase Can Generate Sufficient Phosphoribosyl Amine for Thiamine Synthesis in Salmonella enterica

Abstract

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.Metabolic processes in a living cell are connected through the integration of biochemical pathways. This metabolic integration allows bacteria to survive under diverse conditions and is essential for their adaptation to changing environments. The structure and function of networks created by the integration and overlap of metabolic processes are largely uncharacterized. The thiamine biosynthetic pathway in Salmonella enterica has direct biochemical connections with other metabolic pathways, including purine and isoprenoid biosynthesis (5,33,40), and indirect connections with a number of other cellular processes (1,16,19,25,36). Thus, this biosynthetic pathway serves as a focal point for studies to identify and characterize metabolic interactions, redundancy, and regulation that are essential in a robust metabolism.

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

“…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). UK114/hp14.5 cDNA was provided by G. Schmitz (20).…”

“…Protein concentration was determined with a BCA protein assay kit (Thermo Scientific). Protein activity was verified using a phosphoribosyltransferase assay, as described previously (19,24,25).…”

Mutations in the Tryptophan Operon Allow PurF-Independent Thiamine Synthesis by Altering Flux In Vivo

“…We have hypothesized that PRA synthesis in the absence of PurF is the result of promiscuous enzymes involved in different metabolic processes. This hypothesis was supported by the demonstration that in at least two situations the anthranilate synthase-phosphoribosyltransferase (AS-PRT) complex is required for PRA formation in vivo (4,27). The AS-PRT complex is a multifunctional enzyme (chorismate pyruvate-lyase, EC 4.1.3.27, and N-(5Ј-phosphoribosyl)anthranilate [PR-anthranilate] pyrophosphate phosphoribosyltransferase, EC 2.4.2.18) that catalyzes the first and second steps of tryptophan biosynthesis (2,34) (Fig.…”

“…The reactions were performed in 50 mM potassium phosphate buffer (pH 8.0) in the presence of 10 mM PRPP, 6 mM Mg(Ac) 2 , 2.5 mM ATP, 25 mM [ 14 C]glycine (26 nCi), 10 mM NH 4 Cl, 2 g of GAR synthetase. PurD was overexpressed and purified as previously described (27). In a standard assay, reactions were started by the addition of 5 to 10 g of purified TrpD, followed by incubation at 37°C for 1 h. Various changes to this protocol are noted in the text as relevant for each experiment.…”

Plasticity in the Purine–Thiamine Metabolic Network of Salmonella

“…Mutants of S. enterica lacking amidophosphoribosyltransferase (PurF, EC 2.4.2.14) are able to grow in the absence of thiamine by using alternative mechanisms to generate 5-phosphoribosylamine (PRA), the product of the PurF reaction (Petersen et al 1996;Enos-Berlage et al 1998;Ramos and Downs 2003;Ramos et al 2008;. In each of the described cases, synthesis of thiamine required the subsequent four purine biosynthetic enzymes (PurD, -T, -G, -I).…”