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Journal of Bacteriology, May 2003, p. 2793-2801, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2793-2801.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Escherichia coli phnN, Encoding Ribose 1,5-Bisphosphokinase Activity (Phosphoribosyl Diphosphate Forming): Dual Role in Phosphonate Degradation and NAD Biosynthesis Pathways

Bjarne Hove-Jensen,1* Tina J. Rosenkrantz,1 Andreas Haldimann,2,{dagger} and Barry L. Wanner2

Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark,1 Department of Biology, Purdue University, West Lafayette, Indiana 479072

Received 15 July 2002/ Accepted 3 February 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 + Pi -> 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.

* Corresponding author. Mailing address: Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, 83H Sølvgade, DK-1307 Copenhagen K, Denmark. Phone: 45 3532 2027. Fax: 45 3532 2040. E-mail: hove{at}mermaid.molbio.ku.dk.

{dagger} Present address: Arpida AG, 4142 Münchenstein, Switzerland.

Journal of Bacteriology, May 2003, p. 2793-2801, Vol. 185, No. 9
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.9.2793-2801.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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