Glutamine Phosphoribosylpyrophosphate Amidotransferase-independent Phosphoribosyl Amine Synthesis from Ribose 5-Phosphate and Glutamine or Asparagine

Mark J Koenigsknecht,Itzel Ramos,Diana M Downs

doi:10.1074/jbc.m704024200

Mark J Koenigsknecht, Itzel Ramos + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m704024200

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Abstract

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic l-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed.

Highlights

PurF are able to grow in the absence of thiamine [5, 6]
Initial screens for PRA-forming activity were preformed in S. enterica and E. coli cell-free extracts lacking glutamine-PRPP amidotransferase
Because PRA can be chemically synthesized from ribose 5-phosphate (R5P) and ammonia, this nonenzymatic formation was attributed to small amounts of ammonia in the buffer [15, 19]

Summary

EXPERIMENTAL PROCEDURES

Medium and Chemicals—Culture media supplies were obtained from Difco. Glutamine, asparagine, glycine, ribose 5-phosphate, PRPP, magnesium acetate, ammonium chloride, ATP, EDTA, ␤-mercaptoethanol, phenylmethanesulfonyl fluoride, protamine sulfate, DNase I (EC 3.1.21.1), and lysozyme (EC 3.2.1.17) were obtained from Sigma-Aldrich. The column was washed with two column volumes (CV) of buffer A, and all PRA-forming activity was found to be in the flow-through and wash fractions These fractions were pooled and concentrated using a 30-kDa cut-off Amicon ultrafiltration membrane. The concentrated fraction was applied at a flow rate of 0.5 ml/min to a HiLoad Superdex 75 size exclusion column (24-ml CV) equilibrated with buffer A containing 100 mM KCl; 1-ml fractions were collected and assayed for PRAforming activity. The resin was washed with an additional 200 ml of buffer A, which was pooled with the 200-ml supernatant and concentrated using a using a 30-kDa cut-off Amicon ultrafiltration membrane and applied to a Superdex 75 size exclusion column (see above). A 6-ml wash was combined with the flow-through fraction and concentrated with a 30-kDa cut-off Amicon ultrafiltration membrane

RESULTS

32 Interconversion of L-malate and oxaloacetate talA DM9652 Transaldolase A

DISCUSSION