Induction of phosphoribomutase in Bacillus cereus growing on nucleosides

In this paper we show that phosphoribomutase is induced in Bacillus cereus by the same metabolizable purine and pyrimidine ribonucleosides previously shown to induce the purine nucleoside phosphorylase (Tozzi, M.G., Sgarrella, F. and Ipata, P.L. (1981) Biochim. Biophys. Acta 678, 460–466). The mutase allows ribose 1-phosphate formed from nucleosides to be utilized by the cell through the pentose cycle, upon transformation to ribose 5-phosphate. The equilibrium constant of the mutase reaction is towards ribose-5-phosphate formation. The coordinate induction of the two enzymes completes the picture of the molecular events leading to the utilization of the sugar moiety of purine nucleosides and nucleosides as an energy source (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905–7909).     

Induction and repression of enzymes involved in exogenous purine compound utilization of Bacillus cereus

5'-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine, nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Induction and repression of enzymes involved in exogenous purine compound utilization in Bacillus cereus

5′-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous purine compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented at during growth of B. cereus in the presence of AMP, the concerted action of 5′-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B cereus acts as a translocase of the ribose moiety of ionsine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905–7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Purine and pyrimidine salvage in whole rat brain. Utilization of ATP-derived ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate generated in experiments with dialyzed cell-free extracts.

The object of this work stems from our previous studies on the mechanisms responsible of ribose-1-phosphate- and 5-phosphoribosyl-1-pyrophosphate-mediated nucleobase salvage and 5-fluorouracil activation in rat brain (Mascia, L., Cappiello M., Cherri, S., and Ipata, P. L. (2000) Biochim. Biophys. Acta 1474, 70-74; Mascia, L., Cotrufo, T., Cappiello, M., and Ipata, P. L. (1999) Biochim. Biophys. Acta 1472, 93-98). Here we show that when ATP at "physiological concentration" is added to dialyzed extracts of rat brain in the presence of natural nucleobases or 5-fluorouracil, adenine-, hypoxanthine-, guanine-, uracil-, and 5-fluorouracil-ribonucleotides are synthesized. The molecular mechanism of this peculiar nucleotide synthesis relies on the capacity of rat brain to salvage purine and pyrimidine bases by deriving ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate from ATP even in the absence of added pentose or pentose phosphates. The levels of the two sugar phosphates formed are compatible with those of synthesized nucleotides. We propose that the ATP-mediated 5-phosphoribosyl-1-pyrophosphate synthesis occurs through the action of purine nucleoside phosphorylase, phosphopentomutase, and 5-phosphoribosyl-1-pyrophosphate synthetase. Furthering our previous observations on the effect of ATP in the 5-phosphoribosyl-1-pyrophosphate-mediated 5-fluorouracil activation in rat liver (Mascia, L., and Ipata, P. L. (2001) Biochem. Pharmacol. 62, 213-218), we now show that the ratio [5-phosphoribosyl-1-pyrophosphate]/[ATP] plays a major role in modulating adenine salvage in rat brain. On the basis of our in vitro results, we suggest that massive ATP degradation, as it occurs in brain during ischemia, might lead to an increase of the intracellular 5-phosphoribosyl-1-pyrophosphate and ribose-1-phosphate pools, to be utilized for nucleotide resynthesis during reperfusion.     

Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase

Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2'-deoxypurine nucleosides to the free base and ribose (or 2'-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4'- and 5'-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.     

In vitro recycling of alpha-D-ribose 1-phosphate for the salvage of purine bases

In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273-281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.     

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17β

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.     

Role of nucleosides, 5-phosphoribosyl-1-pyrophosphate, and ribose 1-phosphate in the biosynthesis of phosphosphingolipids in Bacteroides melaninogenicus.

Using a deenergized spheroplast system from Bacterioides melaninogenicus, the sphingolipid precursor 3-ketodihydrosphingosine is not incorporated into the complete sphingolipids, ceramide phosphorylethanolamine, or ceramide phosphorylglycerol unless supplied with glutamine, ATP, ADP, or AMP. Adenosine, inosine, and certain other nucleosides were as effective as ATP. Purine bases and ribose, however, were inactive in this system. 5-Phosphoribosyl-1-pyrophosphate and ribose 1-phosphate also stimulated conversion of 3-ketodihydrosphingosine into ceramide phosphorylethanolamine and ceramide phosphorylglycerol, whereas ribose 5-phosphate showed only slight activity. Hypoxanthine was the main product formed from inosine and adenosine but there was no evidence for nucleotide formation. Adenosine stimulated³²P_i incorporation into cell phospholipids indicating that ribose 1-phosphate, formed via purine nucleoside phosphorylase, could be the compound stimulating sphingolipid synthesis in this system.     

Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

From an Escherichia coli purine auxotroph a mutant defective in phosphoribosylpyrophosphate (PRib-PP) synthetase has been isolated and partially characterized. In contrast to the parental strain, the mutant was able to grow on nucleosides as purine source, whereas growth on purine bases was reduced. Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 muM respectively, compared to 60 muM and 45 muM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate, stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib-PP synthetase activity was observed in both strains, although to a lesser extent in the mutant. Our data suggest that the mutant harbors a mutation in the structural gene for PRib-PP synthetase. The mutation responsible for the altered PRib-PP synthetase was located in the purB-hemA region at 26 min on the recalibrated linkage map.     

Metabolisms of Nucleosides in Bacteria

The mechanism of trans-N-ribosylation in Corynebacterium sepedonicum was investigated. Using the DEAE-cellulose colum chromatography, this enzyme activity was divided into two fractions. One cleaved uridine to uracil and ribose phosphate, and the other decomposed inosine into hypoxanthine and ribose phosphate, in the presence of inorganic phosphate. The ribose phosphate was isolated and crystallized.

Several analytical data indicated that the ribose phosphate was ribose-1-phosphate. These two enzyme fractions catalyzed the formation of nucleosides from ribose-1-phosphate and bases.

Most of bacteria, which had the activity to transfer N-ribosyl group between purine and pyrimidine, could synthesize the nucleoside from base and ribose-1-phosphate.     

Characterization and chemical properties of phosphoribosylamine, an unstable intermediate in the de novo purine biosynthetic pathway

Incubation of [1-13C]-5-phosphoribosyl pyrophosphate ([1-13C]PRPP) and glutamine with PRPP amidotransferase results in rapid production and disappearance of two new resonances at 89.3 and 85.9 ppm. These resonances coincide with two of the products produced upon incubation of [1-13C]ribose 5-phosphate with NH3. Extensive NMR studies (15N and 1H-13C chemical shift correlation spectra) have allowed assignment of these resonances to beta- and alpha-phosphoribosylamine. These studies represent the first spectral observations of this chemically reactive intermediate. The rate of interconversion of alpha- to beta-phosphoribosylamine as a function of pH has been determined by saturation and inversion-transfer NMR methods. The rate of formation of 5-phosphoribosylamine (PRA) from ribose 5-phosphate and NH3 and its rate of decomposition as a function of pH have been determined with a glycinamide ribonucleotide synthetase trapping system fashioned after earlier studies of Nierlich and Magasanik [Nierlich, D. P., & Magasanik, B. (1965) J. Biol. Chem. 240, 366]. Phosphoribosylamine has a t1/2 = 38 s at 37 degrees C and pH 7.5. The pH-independent equilibrium constant for ribose 5-phosphate and NH3 with phosphoribosylamine has been established, 2.5 M-1, by use of these rate constants as well as by NMR methods. This equilibrium constant and the rates of nonenzymatic interconversion of alpha- and beta-PRA provide essential background for studying the mechanism of glycinamide ribonucleotide synthetase and investigating the possibility of channeling phosphoribosylamine between this enzyme and the first enzyme in the purine pathway.     

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.     

Acceleration of purine synthesis in mouse liver by glycogenolytic hormones.

Administration (ip) into fed mice of glucagon, epinephrine, vasopressin, oxytocin, angiotensin II, and dibutyryl cyclic AMP (dbcAMP) resulted in a rapid (within 2.5 to 15 min) elevation of PRPP content (two- to threefold) and in acceleration of the rate of de novo purine synthesis (twofold). Inhibition of the epinephrine-stimulated glycogenolysis by 2,5-anhydromannitol diminished markedly the acceleration effect of the hormone on the rate of purine synthesis. Administration of the hormones caused a rapid rise in the liver content of glucose 6-phosphate (G6P) by 15-70% but did not increase the ribose 5-phosphate (R5P) content. Liver ATP content was not affected. The hormones did not cause direct activation of PRPP synthetase, as gauged by the specific activity of the enzyme, its Km for substrates R5P and ATP, and its sensitivity to inhibition by ADP and GDP. The hormones did not increase the liver content of the enzyme activators Pi and Mg2+. The results suggest that the glycogenolytic hormones accelerate purine synthesis by a metabolic mechanism associated with the enhancement of glycogenolysis. PRPP synthesis is probably enhanced by the glycogenolysis-induced alterations in the cellular content of some metabolites other than R5P.     

Unusual conformational flexibility in N1-substituted uncommon purine nucleosides. Crystal structure of 1-allyl-isoguanosine and 1-allyl-xanthosine.

Several new N1-substituted uncommon purine nucleosides, including doridosine (1-methyl-isoguanosine; m-iG), 1-allyl-isoguanosine (a-iG) and 1-allyl-xanthosine (a-X), have been synthesized and tested as agonists for the adenosine receptors. Some have smooth muscle relaxant or negative chronotropic activities. The X-ray crystal structure of these compounds has been determined at atomic resolution in order to understand the structure-activity relationship. The structures were solved by direct methods and refined by full-matrix least-squares refinement procedure. The crystallographic parameters are: a-iG, space group P2(1), a = 10.573 (1) A, b = 21.955 (2) A, c = 14.360 (1) A, beta = 110.65 (1) degree, no. of 3 sigma Fo's = 4585, R = 0.047; a-X, space group P2(1)2(1)2(1), a = 16.015 (2) A, b = 16.239 (1) A, (1) A, c = 5.3723 (5) A, no. of 3 sigma Fo's = 1169, R = 0.031. In the a-iG crystal, there are 4 independent molecules (with different conformation) per asymmetric unit. While all 4 molecules adopt anti chi CN glycosyl torsion angle, their riboses have 3 distinct puckers (C2'-exo, C2'-endo and C1'-exo). In contrast, the a-X structure adopts a syn chi CN glycosyl torsion angle, which is stabilized by an intramolecular hydrogen bond between the N3 of purine base and the O5' of the ribose (in C2'-endo pucker). Both purine bases (a-iG and a-X) are mainly in the keto tautomer form. For the isoguanine base, the averaged N1-C2 bond distance (1.42 A) is significantly longer than that (1.375 A) of the guanine base. For the xanthine base, N3 nitrogen has an imino proton attached which is unambiguously located in the electron density map. The surprising flexibility in the ribose ring of these N1-substituted uncommon purine nucleosides suggests that the ribose moiety may not participate in the binding of nucleoside to the adenosine receptors.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.     

Phosphoribosylpyrophosphate synthesis from glucose decreases during amino acid starvation of human lymphoblasts

When cultured human lymphoblasts are starved 3 h for an essential amino acid, rates of purine nucleotide synthesis decrease markedly because of a decrease in the intracellular phosphoribosylpyrophosphate concentration (Boss, G.R., and Erbe, R.W. (1982) J. Biol. Chem. 257, 4242-4247; Boss, G. R. (1984) J. Biol. Chem. 259, 2936-2941). In amino acid-starved cells, glucose transport was not changed, whereas total glucose consumption and lactate production decreased by approximately 25 and 10%, respectively. Carbon flow through the oxidative pentose phosphate pathway, measured by 14CO2 release from [1-14C]glucose, decreased by 18% during amino acid starvation. However, kinetic studies of ribulose-5-phosphate 3-epimerase and phosphoriboisomerase suggested that the ribulose 5-phosphate produced by this pathway is converted mostly to xylulose 5-phosphate instead of to ribose 5-phosphate so that this pathway produces little phosphoribosylpyrophosphate. The activity of the nonoxidative pentose phosphate pathway, measured by high performance liquid chromatography following the incorporation of [1-14C]glucose into phosphoribosylpyrophosphate, ATP, and GTP, decreased by approximately 55% during amino acid starvation. None of the enzymes of either pathway changed in specific activity during amino acid starvation. We conclude that the nonoxidative pentose phosphate pathway is the major source of phosphoribosylpyrophosphate for purine nucleotide synthesis and that this pathway is regulated by a metabolite which changes in concentration during amino acid starvation.     

Pentose phosphates in nucleoside interconversion and catabolism

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.     

Mechanism of Action of Showdomycin

¹⁴C-Labelled showdomycin was rapidly taken up by Escherichia coli K-12 cells. The showdomycin uptake was highly temperature dependent, sensitive to azide and N-ethyl-maleimide, but was only partially inhibited by treatment with high concentration of iodoacetic acid.

The uptake of showdomycin was inhibited by a wide variety of nucleosides but not by purine and pyrimidine bases, nucleotides, ribose or ribose-5-phosphate. The inhibition of showdomycin uptake by adenosine was of a competitive type.

Since nucleosides inhibited the uptake of showdomycin but did not facilitate its efflux, they must play a role of inhibitors to the entry of the antibiotic into cells.

Removal of extracellular showdomycin by washing, or inhibition of its subsequent entry into cells by the addition of nucleosides or sulfhydryl compounds resulted in a rapid decrease in the intracellular level of the antibiotic during subsequent incubation.     

Autophosphorylation of phosphoglucosamine mutase from Escherichia coli

Phosphoglucosamine mutase (GlmM) catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, an essential step in the pathway for UDP-N-acetylglucosamine biosynthesis in bacteria. This enzyme must be phosphorylated to be active and acts according to a ping-pong mechanism involving glucosamine-1, 6-diphosphate as an intermediate (L. Jolly, P. Ferrari, D. Blanot, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx, Eur. J. Biochem. 262:202-210, 1999). However, the process by which the initial phosphorylation of the enzyme is achieved in vivo remains unknown. Here we show that the phosphoglucosamine mutase from Escherichia coli autophosphorylates in vitro in the presence of [(32)P]ATP. The same is observed with phosphoglucosamine mutases from other bacterial species, yeast N-acetylglucosamine-phosphate mutase, and rabbit muscle phosphoglucomutase. Labeling of the E. coli GlmM enzyme with [(32)P]ATP requires the presence of a divalent cation, and the label is subsequently lost when the enzyme is incubated with either of its substrates. Analysis of enzyme phosphorylation by high-pressure liquid chromatography and coupled mass spectrometry confirms that only one phosphate has been covalently linked to the enzyme. Only phosphoserine could be detected after acid hydrolysis of the labeled protein, and site-directed mutagenesis of serine residues located in or near the active site identifies the serine residue at position 102 as the site of autophosphorylation of E. coli GlmM.