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.

FIG. 1.

Inputs to the synthesis of PRA in S. enterica. Schematically represented are the purine, thiamine, and tryptophan biosynthetic pathways. When present, gene names are indicated by the biosynthetic step catalyzed by their product. The dotted lines represent the pathways proposed to contribute to PRA synthesis in the absence of the PurF enzyme. PR-anthranilate, phosphoribosyl anthranilate; PPi, pyrophosphate; R5P, ribose-5-phosphate; AIR, aminoimidazole ribotide; THZ-P, 4-methyl-5-(β-hydroxyethyl)-thiazole phosphate; HMP-PP, 4-amino-5-hydroxymethyl-2-methyl pyrimidine pyrophosphate; TPP, thiamine pyrophosphate.

FIG. 2.

TrpDE- and PurF-catalyzed reactions show biochemical similarity. Biochemical reactions catalyzed by the AS-PRT complex (TrpDE) and glutamine phosphoribosyl amidotransferase (PurF) are illustrated to emphasize similarities. Each enzyme has a glutaminase activity and transfers a phosphoribosyl group to an amidated substrate.

FIG.3.

TrpDE, provided in trans, allows thiamine-independent growth. Growth analyses were preformed at 37°C as described in Materials and Methods. Growth of DM6808 [purF2085 gnd-181(pSU19)] (▪), DM6809 [purF2085 gnd-181(pIR-EDW)] (▵), DM6810 [purF2085 gnd-181(pIR-EDMS)] (▴), DM6811 [purF2085 gnd-181(pIR-DW)] (○), and DM6812 [purF2085 gnd-181(pIR-DMS)] (•) in glucose-adenine medium is shown.

FIG. 4.

Synthesis of PRA from PRPP and NH₄Cl. PRA-forming activity in a cell extract from strain DM6417 was determined by using a coupled assay with GAR synthetase. In the coupled reaction a molecule of provided [¹⁴C]glycine is incorporated into the PRA structure. Synthesis of PRA from PRPP and NH₄Cl was determined as a function of [¹⁴C]GAR synthesis. Labeled GAR and glycine were separated on polyethyleneimine-cellulose with a methanol-pyridine-water (20:1:5) solvent system. Quantification determined that lane 3 contained twofold more GAR than the control lane without NH₄Cl. No increase in GAR formation was seen with cell extracts from DM6418.

FIG. 5.

Schematic representation of wild-type and mutant AS-PRT complexes. AS-PRT catalyzes the first two steps in tryptophan biosynthesis, which involve amidotransferase and PRT activities of component II (TrpD). The site for allosteric inhibition by tryptophan is on TrpE, and its approximate location is indicated. Solid lines represent the defined reaction path. Dotted lines reflect the proposed reaction catalyzed by the AS complex leading to PRA synthesis. An asterisk indicates the position of the trpD3611 mutation in TrpD.