Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene

Andersen, Paal Skytt; Frees, Dorte; Fast, R; Mygind, Bente

doi:10.1128/jb.177.8.2008-2013.1995

Cited by 59 publications

(60 citation statements)

References 37 publications

(46 reference statements)

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“…So, unless the concentration of divalent metal ions is limiting inside the cells, the mechanism of inhibition of uracil uptake under stringent conditions is still unknown. Possible explanations for the slow incorporation of uracil include a drop in the intracellular concentration of PPRibP during the stringent response, or inhibition of the uracil transport system by ppGpp [47], but it is also likely that the reduced consumption of nucleotides for stable RNA synthesis during the stringent response by itself reduces the turnover of nucleotide pools and thereby delays incorporation of exogenous uracil.…”

Section: Discussionmentioning

confidence: 99%

Different Oligomeric States are Involved in the Allosteric Behavior of Uracil Phosphoribosyltransferase from Escherichia Coli

Jensen

Mygind

1996

European Journal of Biochemistry

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Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl-a-I-diphosphate (PPRibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing K,, for PPRibP by about fivefold without affecting V,,,,,. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PPRibP displayed constant velocities. This indicated that PPRibP and GTP influenced the structure of the enzyme. Gel-filtration and sedimentation analyses showed that the apparent oligomeric state of uracil phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active. It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PPRibP. Guanosine-3',5'-bis(diphosphate) was found to activate the enzyme much like GTP.Keywoi-ds: pyrimidine biosynthesis ; pyrimidine salvage; UMP; GTP activation ; 5-phosphoribosyl 1 -diphosphate ; stringent response.The phosphoribosyltransferases catalyse the formation of nucleotides and pyrophosphate from 5-phosphoribosyl-a-I -diphosphate (PPRibP) and a nitrogenous base which usually is aromatic. There are 10 different phosphoribosyltransferases in Escherichia coli. They participate in the biosynthesis of purine and pyrimidine nucleotides, pyridine coenzymes, and the amino acids histidine and tryptophan. Two of the phosphoribosyltransferases participate in pyrimidine nucleotide synthesis, namely (a) orotate phosphoribosyltransferase (OPRT) which catalyses a step in the de novo pathway for pyrimidine nucleotide synthesis and (b) uracil phosphoribosyltransferase (UPRT) which is responsible for salvage of preformed uracil [l].OPRT has been characterised extensively and the three-dimensional structure of the protein is known [2, 31. The enzyme is a stable dimer of identical polypeptides [4-71 with two shared active sites present in the interface between the subunits of the dimer, so that amino acid residues from both subunits contribute to catalysis [2, 3, 6, 81. UPRT was discovered by Kornberg and his collaborators during their pioneering work resolving the pathways of nucleotide biosynthesis [9]. The enzyme from E. coli is stimulated by GTP by an unknown mechanism [10][11][12]. This property makes UPRT functionally distinct from OPRT, which does not seem to be regulated by the same mechanism. Moreover, it was reported that UPRT is inhibited by the regulatory nucleotide, guanosine 3',5'-bis(diphosphate) ppGpp [I I ] that accumulates in E. coli under starvation conditions [13].We report here on the GTP and PPRibP modulation of UPRT quaternary structure.

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Section: Discussionmentioning

confidence: 99%

Different Oligomeric States are Involved in the Allosteric Behavior of Uracil Phosphoribosyltransferase from Escherichia Coli

Jensen

Mygind

1996

European Journal of Biochemistry

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“…Briefly, wild type PhnP with a C-terminal His 6 tag was expressed from the plasmid pHO520-phnP in BL21(DE3) cells (Novagen) co-transformed with pLacI (Novagen). The plasmid pHO520-phnP is a derivative of pUHE 23-2 which contains T7 promoter and lac operator sites (14). Cells were grown in LB medium supplemented with amplicillin and chloramphenicol at 30°C and induced at A 600 ϳ 0.6 with 0.5 mM isopropyl-␤-D-1-thiogalactopyranoside.…”

Section: Methodsmentioning

confidence: 99%

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Podzelinska

Wathier

et al. 2009

Journal of Biological Chemistry

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Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the ␤-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(pnitrophenyl)phosphate and 2,3-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn 2؉ and Ni 2؉ for catalysis, whereas Zn 2؉ afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 Å resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn 2؉ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn 2؉ binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.

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“…Although no nucleobase transporter has been identified in mammals, several nucleobase transporters have been identified and well characterized in lower organisms, such as bacteria (18), fungi (19 -22), and protozoa (23,24), and in plants (25,26). They have been categorized into three families of nucleobaseascorbate transporter (NAT), microbial purine-related transporter, and plant purine-related transporter, based on their molecular and functional characteristics (4).…”

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confidence: 99%

Identification and Functional Characterization of the First Nucleobase Transporter in Mammals

Yamamoto

Inoue

Murata

et al. 2010

Journal of Biological Chemistry

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Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodiumdependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 M. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.Nucleosides are essential components of DNA and RNA, the genomic memory devices, and also play pivotal roles as energy sources and in signal transductions (1, 2). In mammals, nucleosides can be synthesized via the de novo pathway from small precursor molecules, such as aspartate, glutamine, glycine, and 10-formyltetrahydrofolate, and via the salvage pathway from nucleobases. In the salvage pathway, the supply of nucleobases mainly occurs from extracellular sources, typically those produced by digestion of dietary nucleic acids in the intestinal lumen. There also occurs redistribution of nucleosides/nucleobases via bloodstream from the tissues that are highly active in de novo synthesizing and/or degrading nucleosides. Therefore, carrier-mediated transport systems are needed for the efficient trafficking across cellular membranes of nucleosides and nucleobases, which are hydrophilic and hence can little permeate otherwise (3, 4).The transporters involved in the cellular uptake of nucleosides have been well characterized in mammals, including humans (5-7). There are two families of transporters, which are sodium-coupled concentrative nucleoside transporters (CNT1/SLC28A1, CNT2/SLC28A2, and CNT3/SLC28A3) and equilibrative nucleoside transporters (ENT1/SLC29A1 and ENT2/SLC29A2...

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Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene

Cited by 59 publications

References 37 publications

Different Oligomeric States are Involved in the Allosteric Behavior of Uracil Phosphoribosyltransferase from Escherichia Coli

Different Oligomeric States are Involved in the Allosteric Behavior of Uracil Phosphoribosyltransferase from Escherichia Coli

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

Identification and Functional Characterization of the First Nucleobase Transporter in Mammals

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