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J. Biol. Chem., Vol. 282, Issue 39, 28379-28384, September 28, 2007

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

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

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 2.4.2.1 [EC] 4) 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 [EC] ), 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 [EC] ), 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.

Received for publication, May 16, 2007 , and in revised form, July 23, 2007.

^* This work was supported in part by National Institutes of Health Grant GM47296. Funds were also provided from a 21st Century Scientist Scholars Award from the J. S. McDonnell Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² Supported by a pre-doctoral fellowship from Consejo Nacional de Ciencia y Tecnologia (Mexico). Present address: Depts. of Biology and Earth and Planetary Sciences, M.I.T., Cambridge, MA 02139.

³ To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin, 1550 Linden Dr., Madison, WI 53706. Tel.: 608-265-4630; Fax: 608-262-9865; E-mail: downs{at}bact.wisc.edu.

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