Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways

J Bacteriol. 2003 May;185(9):2793-801. doi: 10.1128/JB.185.9.2793-2801.2003.


An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene encoding PRPP synthase (B. Hove-Jensen, J. Bacteriol. 178:714-722, 1996). The new pathway requires three enzymes: phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase. The latter activity is encoded by phnN; the product of this gene is required for phosphonate degradation, but its enzymatic activity has not been determined previously. The reaction sequence is ribose 5-phosphate --> ribose 1-phosphate --> ribose 1,5-bisphosphate --> PRPP. Alternatively, the synthesis of ribose 1-phosphate in the first step, catalyzed by phosphopentomutase, can proceed via phosphorolysis of a nucleoside, as follows: guanosine + P(i) --> guanine + ribose 1-phosphate. The ribose 1,5-bisphosphokinase-catalyzed phosphorylation of ribose 1,5-bisphosphate is a novel reaction and represents the first assignment of a specific chemical reaction to a polypeptide required for cleavage of a carbon-phosphorus (C-P) bond by a C-P lyase. The phnN gene was manipulated in vitro to encode a variant of ribose 1,5-bisphosphokinase with a tail consisting of six histidine residues at the carboxy-terminal end. PhnN was purified almost to homogeneity and characterized. The enzyme accepted ATP but not GTP as a phosphoryl donor, and it used ribose 1,5-bisphosphate but not ribose, ribose 1-phosphate, or ribose 5-phosphate as a phosphoryl acceptor. The identity of the reaction product as PRPP was confirmed by coupling the ribose 1,5-bisphosphokinase activity to the activity of xanthine phosphoribosyltransferase in the presence of xanthine, which resulted in the formation of 5'-XMP, and by cochromatography of the reaction product with authentic PRPP.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Lyases / chemistry
  • Lyases / metabolism*
  • NAD / biosynthesis
  • NAD / metabolism*
  • Pentosephosphates / metabolism
  • Phosphoribosyl Pyrophosphate / biosynthesis*
  • Phosphotransferases / metabolism
  • Ribose-Phosphate Pyrophosphokinase / metabolism*
  • Ribosemonophosphates / metabolism


  • Pentosephosphates
  • Ribosemonophosphates
  • NAD
  • ribose-1,5-bisphosphate
  • ribose-5-phosphate
  • Phosphoribosyl Pyrophosphate
  • Adenosine Triphosphate
  • Phosphotransferases
  • Ribose-Phosphate Pyrophosphokinase
  • Lyases
  • carbon-phosphorus lyase
  • phosphopentomutase