SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

SUMMARY After several decades of use of glyphosate, the active ingredient in weed killers such as Roundup, in fields, forests, and gardens, the biochemical pathway of transformation of glyphosate phosphorus to a useful phosphorus source for microorganisms has been disclosed. Glyphosate is a member of a large group of chemicals, phosphonic acids or phosphonates, which are characterized by a carbon-phosphorus bond. This is in contrast to the general phosphorus compounds utilized and metabolized by microorganisms. Here phosphorus is found as phosphoric acid or phosphate ion, phosphoric acid esters, or phosphoric acid anhydrides. The latter compounds contain phosphorus that is bound only to oxygen. Hydrolytic, oxidative, and radical-based mechanisms for carbon-phosphorus bond cleavage have been described. This review deals with the radical-based mechanism employed by the carbon-phosphorus lyase of the carbon-phosphorus lyase pathway, which involves reactions for activation of phosphonate, carbon-phosphorus bond cleavage, and further chemical transformation before a useful phosphate ion is generated in a series of seven or eight enzyme-catalyzed reactions. The phn genes, encoding the enzymes for this pathway, are widespread among bacterial species. The processes are described with emphasis on glyphosate as a substrate. Additionally, the catabolism of glyphosate is intimately connected with that of aminomethylphosphonate, which is also treated in this review. Results of physiological and genetic analyses are combined with those of bioinformatics analyses.

Escherichia coli strains defective in the rpiA gene, encoding ribose phosphate isomerase A, are ribose auxotrophs, despite the presence of the wild-type rpiB gene, which encodes ribose phosphate isomerase B. Ribose prototrophs of an rpiA genetic background were isolated by two different approaches. Firstly, spontaneous ribose-independent mutants were isolated. The locus for this lesion, rpiR, was mapped to 93 min on the linkage map, and the gene order zje::Tn10-rpiR-mel-zjd::Tn10-psd-purA was established. Secondly, ribose prototrophs resulted from the cloning of the rpiB gene on a multicopy plasmid. The rpiB gene resided on a 4.6-kbp HindIII-EcoRV DNA fragment from phage 10H5(642) of the Kohara gene library and mapped at 92.85 min. Consistent with this map position, the cloned DNA fragment contained two divergent open reading frames of 149 and 296 codons, encoding ribose phosphate isomerase B (molecular mass, 16,063 Da) and a negative regulator of rpiB gene expression, RpiR (molecular mass, 32,341 Da), respectively. The 5 ends of rpiB-and rpiR-specified transcripts were located by primer extension analysis. No significant amino acid sequence similarity was found between ribose phosphate isomerases A and B, but ribose phosphate isomerase B exhibited high-level similarity to both LacA and LacB subunits of the galactose 6-phosphate isomerases of several gram-positive bacteria. Analyses of strains containing rpiA, rpiB, or rpiA rpiB mutations revealed that both enzymes were equally efficient in catalyzing the isomerization step in either direction and that the construction of rpiA rpiB double mutants was a necessity to fully prevent this reaction.

The sequential activities of PhnY, an α-ketoglutarate/Fe(II)-dependent dioxygenase, and PhnZ, a Fe(II)-dependent enzyme of the histidine-aspartate motif hydrolase family, cleave the carbon-phosphorus bond of the organophosphonate natural product 2-aminoethylphosphonic acid. PhnY adds a hydroxyl group to the α-carbon, yielding 2-amino-1-hydroxyethylphosphonic acid, which is oxidatively converted by PhnZ to inorganic phosphate and glycine. The PhnZ reaction represents a new enzyme mechanism for metabolic cleavage of a carbon-phosphorus bond.

A mutant of Escherichia coli harboring a temperature-labile phosphoribosylpyrophosphate (PRPP) synthetase was characterized. Despite the lack of a detectable PRPP pool or PRPP synthetase activity at 40°C, the strain was fully viable at this temperature as long as guanosine, uridine, histidine, tryptophan, and nicotinamide mononucleotide were all added to the growth medium. Viability of the strain was dependent upon mutations in genes of the nucleoside salvage pathways that improved the utilization of exogenous nucleosides. The properties of the strain are those expected of a PRPP-less strain and suggest that PRPP synthetase is dispensable for E. coli.The metabolite 5-phospho-D-ribosyl-a-1-pyrophosphate (PRPP) is a biosynthetic precursor of purine and pyrimidine nucleotides, the pyridine nucleotide coenzyme NAD, and the amino acids histidine and tryptophan (8). In nucleotide synthesis, PRPP is used in the de novo pathways as well as in the auxiliary pathways by which bases are converted to the nucleotides. Thus, in Escherichia coli 10 enzymes utilize PRPP as a substrate (Fig. 1A). The synthesis of PRPP is catalyzed by PRPP synthetase (ATP:D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) as follows: ribose 5-phosphate + ATP --PRPP + AMP. This enzyme, encoded by the prs gene (4-6), is believed to be essential for the growth of all organisms. The ultimate products of some of the PRPP pathways, i.e., purine and pyrimidine nucleotides, are impermeable to cells and therefore cannot be fed exogenously. Instead, in E. coli wild-type cells the nucleotides and nucleosides are catabolized rapidly to the nucleobases, and the nucleobases in turn are converted intracellularly to the nucleotides by consumption of PRPP (Fig. 1B) (3,12,14). Moreover E. coli cells are impermeable to PRPP. In the present work, I describe conditions under which PRPP is apparently dispensable. These conditions were achieved by using a strain with a temperature-sensitive mutation within the prs gene and by mutational manipulation of the nucleoside salvage pathways, so that the ribonucleosides were directly phosphorylated to ribonucleotides rather than degraded to nucleobases. As a consequence of these mutations, the strain had a simultaneous requirement for purine, pyrimidine, and pyridine compounds as well as for histidine and tryptophan.MATERIALS AND METHODS Bacterial strains and growth conditions. The E. coli K-12 strains used are shown in Table 1. Standard genetic techniques were used (4). The udp, deoD, and gsk alleles were manipulated in a hemA prs+ host strain which was then transduced to hem' with a bacteriophage P1 lysate grown on H0541 (prs-2) (5), and isogenic hem' prs-2 and hem' prs+ strains were obtained. The markers hemA and prs are very closely linked (4). The manipulation of the markers was performed in a hemA prs+ strain, rather than in a prs-2 strain, to avoid eventual counterselection of prs-2. Strains harboring prs-2 were maintained at 25°C. All udp strains contained the udp::TnS allele originally present in strain AM427 (A...

In Escherichia coli , internalization and catabolism of organophosphonicacids are governed by the 14-cistron phnCDEFGHIJKLMNOP operon. The phnP gene product was previously shown to encode a phosphodiesterase with unusual specificity toward ribonucleoside 2',3'-cyclic phosphates. Furthermore, phnP displays shared synteny with phnN across bacterial phn operons. Here the role of PhnP was examined by (31)P NMR spectrometry on the culture supernatants of E. coli phn mutants grown in the presence of alkylphosphonic acid or phosphite. The addition of any of these alkylphosphonic acids or phosphite resulted in the accumulation of α-D-ribosyl 1,2-cyclic phosphate and α-D-ribosyl 1-alkylphosphonate in a phnP mutant strain. Additionally, α-D-ribosyl 1-ethylphosphonate was observed to accumulate in a phnJ mutant strain when it was fed ethylphosphonic acid. Purified PhnP was shown to regiospecifically convert α-D-ribosyl 1,2-cyclic phosphate to α-D-ribosyl 1-phosphate. Radiolabeling studies revealed that 5-phospho-α-D-ribosyl 1,2-cyclic phosphate also accumulates in a phnP mutant. This compound was synthesized and shown to be regiospecifically converted by PhnP to α-D-ribosyl 1,5-bisphosphate. It is also shown that organophosphonate catabolism is dependent on the synthesis of 5-phospho-α-D-ribosyl 1-diphosphate, suggesting that this phosphoribosyl donor is used to initiate the carbon-phosphorus (CP) lyase pathway. The results show that 5-phospho-α-D-ribosyl 1,2-cyclic phosphate is an intermediate of organophosphonic acid catabolism, and it is proposed that this compound derives from C-P bond cleavage of 5-phospho-α-D-ribosyl 1-alkylphosphonates by CP lyase.

The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(␣)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast twohybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es).The enzyme 5-phosphoribosyl-1(␣)-pyrophosphate synthetase (ATP:D-ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1) (PRS) 1 catalyzes the reaction at a key junction in intermediary metabolism. PRS transfers the pyrophosphate moiety released from ATP to ribose-5-phosphate, thus giving rise to phosphoribosyl-pyrophosphate (PRPP) (1), and the enzyme therefore directs ribose-5-phosphate from energy generated by the pentose phosphate pathway to the important biosynthetic intermediate PRPP. PRPP is a precursor for the production of purine, pyrimidine, and pyridine nucleotides and the amino acids histidine and tryptophan (2). PRPP is required for both the de novo and the salvage pathways of nucleotide metabolism (3). It has been shown that in Mycobacterium spp. PRPP is also required for the biosynthesis of polyprenylphosphate pentoses that contribute to the arabinosyl residues of the cell wall (4).PRS genes have been cloned and sequenced from a variety of organisms; bacteria (5-9), mycoplasma (10), and protozoa (11) each contain apparently one PRS gene. In nematodes (12, 13) and the yeast Schizosaccharomyces pombe (14, 15) two PRS genes have been found so far, whereas in Spinacia oleracea four PRS cDNAs have been identified (16). PRS genes have also been cloned in rat (17-20) and human (21...

SummaryPhosphorous is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use organic phosphonate compounds, which require specialised enzymatic machinery for breaking the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolises phosphonate remain unknown. Here we determine the crystal structure of the 240 kDa Escherichia coli C-P lyase core complex (PhnGHIJ) and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected selfhomologies. It contains two potential active sites that likely couple organic phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy and show that it binds to PhnJ via a conserved insertion domain. Our results provide a structural basis for understanding microbial phosphonate breakdown.Phosphonate compounds that contain a stable carbon-phosphorus (C-P) bond are utilised as a source of phosphate by microorganisms in many natural environments where the low levels of free and organic phosphate limit growth 1 . The C-P lyase pathway, which converts phosphonate into 5-phosphoribosyl-α-1-diphosphate (PRPP) in an ATP-dependent fashion, is activated upon phosphate starvation in many bacterial species including Escherichia coli 2,3 . The enzymes of this pathway have a very broad substrate specificity enabling the Reprints and permissions information is available at www.nature.com/reprintsUsers may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http:// www.nature.com/authors/editorial_policies/license.html#terms 3 Correspondence and requests for materials should be addressed to D.E.B.(deb@mbg.au.dk, phone +45 21669001). Author Contributions. P.S., L.A.P., B.H.J., B.J., and D.E.B. designed and P.S., L.B.V., C.J.R, and B.J. carried out the experiments. P.S., M.K., and D.E.B. determined the crystal and EM structures while C.J.R. and L.A.P. carried out final refinement of the EM structure as well as EM structure validation. P.S, M.K., C.J.R., L.A.P., B.H.J., B.J., and D.E.B. wrote the manuscript.Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) with accession code 4XB6. The EM density map has been deposited in the Electron Microscopy Data Bank (EMDB) with accession code EMD-3033.