Glutamine phosphoribosylpyrophosphate amidotransferase-independent phosphoribosyl amine synthesis from ribose 5-phosphate and glutamine or asparagine.
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.
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706.
Mark J
MJ
Koenigsknecht
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706.
Itzel
I
Ramos
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706. Electronic address: downs@bact.wisc.edu.
Diana M
DM
Downs
D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000582:Amidophosphoribosyltransferase / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000096:biosynthesis* / Q000378:metabolism*; D019779:Salmonella enterica / Q000201:enzymology*
D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D012267:Ribosemonophosphates; D005973:Glutamine; C008891:phosphoribosylamine; C031626:ribose-5-phosphate; D001216:Asparagine; D000582:Amidophosphoribosyltransferase; D000135:Acid Phosphatase; C508128:AphA protein, E coli