Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo

Abstract

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

FIG. 1.

Reactions catalyzed by the TrpE, TrpD, and TrpC enzymes in the tryptophan biosynthetic pathway. (A) The heterotetrameric complex (TrpE₂D₂) catalyzes glutamine-dependent anthranilate synthesis. The second activity, PR-anthranilate synthesis, is performed by the phosphoribosyl transferase subunit (TrpD). Gln, glutamine; Glu, glutamate; Pyr, pyruvate. (B) Reactions catalyzed by the bifunctional enzyme TrpC. The carboxy-terminal domain catalyzes the isomerization of PR-anthranilate to CdRP. The amino-terminal domain catalyzes the decarboxylation of CdRP to indole-3-glycerol-phosphate. PR-Ant, PR-anthranilate; InGP, indole-3-glycerol-phosphate.

FIG. 2.

[¹⁴C]GAR formation in the presence of TrpD enzyme requires anthranilate. (A) Reactions were performed in 50 mM potassium phosphate buffer (pH 8.0) in the presence of 150 μM anthranilate (when added), 10 mM PRPP, 10 mM NH₃, 6 mM Mg(Ac)₂, 2.5 mM ATP, 25 mM [¹⁴C]glycine (26 nCi), and 2 μg of GAR synthetase. Reactions were started by the addition of 5 μg of TrpD enzyme, followed by incubation at 37°C for 1 h. Labeled GAR and glycine were separated on PEI-cellulose by using a methanol-pyridine-water (20:1:5) solvent system. +, addition; −, no addition, with visualization using a Cyclone storage phosphor system. (B) A schematic of the coupled assay used to detect PRA formation is shown.

FIG. 3.

GAR synthesis versus anthranilate concentration. Production of GAR by wild-type TrpD was determined in the presence of increasing concentrations of anthranilate. The assay was performed as described in Materials and Methods in the presence of 10 mM PRPP and 10 mM NH₃. Reactions were started by the addition of 5 μg of the TrpD enzyme, followed by incubation at 37°C for 1 h. The data represent averages for duplicate experiments, and detected [¹⁴C]GAR was quantified in phosphorimager (PI) units.

FIG. 4.

Degradation of PR-anthranilate. The phosphorybosyl transferase activity of TrpD was assayed as described in Materials and Methods in the presence of 1 mM PRPP, 1 mM anthranilate, and 0.3 μg of TrpD enzyme. Disappearance of anthranilate was followed fluorometrically (excitation wavelength, 325 nm; emission, 400 nm) in the presence (○) or absence (□) of TrpD.

FIG. 5.

Nonenzymatic PRA synthesis from NH₃ and R5P in the presence of TrpD. R5P released from the degradation of PR-anthranilate (PR-Ant) formed by TrpD reacts nonenzymatically with NH₃ to produce PRA. Ant., anthranilate; PR-Ant., phosphoribosyl-anthranilate.

FIG. 6.

Metabolite accumulation allows PRA formation in vivo. Strains were grown in minimal medium supplemented with adenine and tryptophan. ▪, DM9784 (purF2085 gnd-181); •, DM9719 (purF2085 gnd-181 ΔtrpR3614 trpE3613 trpC::Tn10). Addition of thiamine restores growth of DM9784 (□). Data shown are averages for two independent cultures, with error bars indicated.

FIG. 7.

Three-dimensional localization of mutations restoring PRA synthesis. The structure of PR-AnI-IGPS (TrpC) (26) is shown. Residue changes are indicated in different colors. The region presumed to be predominately truncated in TrpC(G339Ter) is shown in yellow, starting with residue G339 in pink. The locations of the active sites of the PR-AnI and IGPS domains are indicated by I and S, respectively. Structural depictions were generated using PyMOL software version 0.99 (PyMOL, LLC).

FIG. 8.

Anthranilate excretion by trpC mutants. Feeding experiments were performed as described in Materials and Methods. The indicator strain trpE8 was seeded as an overlay on minimal medium plates supplemented with adenine and thiamine. Indicated mutant strains were stabbed into the agar lawn from overnight cultures. Mutant strains used were DM7870 (purF2085 gnd-181 trpC3626), 7863 (purF2085 gnd-181 trpC3620), 7865 (purF2085 gnd-181 trpC3621), 7869 (purF2085 gnd-181 trpC3624), and 7868 (purF2085 gnd-181 trpC3623). Strain DM9784 (purF2085 gnd-181) is the parental control, and the only growth seen is of the strain itself. Plates were incubated at 37°C for 24 h.