Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways.
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.
Hove-Jensen
Bjarne
B
Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark. hove@mermaid.molbio.ku.dk
Rosenkrantz
Tina J
TJ
Haldimann
Andreas
A
Wanner
Barry L
BL
D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008190:Lyases / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000096:biosynthesis / Q000378:metabolism*; D010428:Pentosephosphates / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate / Q000096:biosynthesis*; D010770:Phosphotransferases / Q000378:metabolism; D012268:Ribose-Phosphate Pyrophosphokinase / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000378:metabolism
D010428:Pentosephosphates; D012267:Ribosemonophosphates; D009243:NAD; C050562:ribose-1,5-bisphosphate; C031626:ribose-5-phosphate; D010754:Phosphoribosyl Pyrophosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D012268:Ribose-Phosphate Pyrophosphokinase; D008190:Lyases; C051328:carbon-phosphorus lyase; C022375:phosphopentomutase