Carlos Eduardo Domenech - Academia.edu (original) (raw)

Papers by Carlos Eduardo Domenech

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium... more Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Bot...

Biology of Reproduction, 1972

Microbiology (Reading, England), 1999

Sterols, carotenoids and gibberellins are synthesized after the reduction of 3-hydroxy-3-methylgl... more Sterols, carotenoids and gibberellins are synthesized after the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate in different subcellular compartments of the fungus Gibberella fujikuroi. Lovastatin inhibits growth in many organisms, presumably because of the inhibition of the synthesis of essential terpenoids. However, in G. fujikuroi growth of the mycelia and sterol and carotenoid content were not affected by the presence of lovastatin. Nevertheless, lovastatin did inhibit the accumulation of gibberellins in the culture medium; this inhibition, however, was counteracted by the addition of mevalonate to the medium. The conversion of HMG-CoA to mevalonate in cell-free extracts was inhibited by 10 nM lovastatin. Since G. fujikuroi apparently possesses a single gene for HMG-CoA reductase, as shown by Southern hybridization and PCR amplification, it was concluded that the biosynthesis of sterols, carotenoids and gibberellins shares a single HMG-CoA reductase, b...

Enzyme Research, 2011

Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, be... more Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it ...

Molecular and Cellular Biochemistry, 1990

Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a peripla... more Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg 2+, the K~ values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K~ ~ values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.

Journal of Bacteriology, 2002

The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain... more The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of P. aeruginosa PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in gbt blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.

FEMS Microbiology Letters, 1999

Gibberellins, fatty acids and the polyketides bikaverin and fusarin C are synthesized from a comm... more Gibberellins, fatty acids and the polyketides bikaverin and fusarin C are synthesized from a common precursor, acetyl-CoA. The production of these compounds in Gibberella fujikuroi was strongly influenced by aeration, determined by the air/medium ratio in shaken batch cultures. Higher aeration resulted in increased growth and the production of bikaverin and gibberellins. Low aeration stimulated fatty acid and fusarin C production. Feeding experiments with labeled leucine or acetate resulted in different proportions of labeled palmitic acid and bikaverin, indicating that these compounds are synthesized from independent acetate pools.

FEMS Microbiology Letters, 1998

Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the pr... more Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively. This system obeys Michaelis-Menten kinetics with an apparent K m value of 53 WM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine.

FEMS Microbiology Letters, 1999

Production of bikaverin and gibberellins by Gibberella fujikuroi started after depletion of the n... more Production of bikaverin and gibberellins by Gibberella fujikuroi started after depletion of the nitrogen source, but not after depletion of phosphate. Despite this similarity, the regulation of both pathways by nitrogen involved two different mechanisms. This conclusion was supported by the fact that the production of bikaverin, in contrast to the gibberellins, was not inhibited by nitrate in a mutant that could not utilize it. The different regulation of both pathways was clearly demonstrated by a mutant that overproduced bikaverin but lacked gibberellins. An optimal bikaverin production required a low pH, with a sharp drop at about pH 5. The syntheses of fungal secondary metabolites, such as bikaverin and gibberellins, are not subject to common regulation, but respond to various combinations of signals, such as nitrogen availability, nitrate and the pH of the medium.

FEMS Microbiology Letters, 1989

FEMS Microbiology Letters, 2006

FEBS Letters, 1992

In Pseudomonas aeruginoSao the effect of different cations on the acid phosphatase activity was s... more In Pseudomonas aeruginoSao the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipas¢ C. Although the natural substrate of this enzyme is phosphoryleholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p.nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorycholine phosphatase) mediated by divalent cations Mg 2÷, Zn ~' ÷ and Cu:* revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Ks, values obtained for p-NPP in the presence of Mg "-+, Zn ~' + and Cu:" were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The ICA values for the same ions were 1.25 raM, 0.05 mM and 0.03 raM0 respectively. The Vm,~ obtained in the presence ofCa :+ was about twofold higher than that obtained in the presence of Mg ~ or Zn-'*. The inhibition observed with AI ~" seems to be a multi-site inhibition. The/c',~p and n values, from the Hill plot, were about 0.25 mM and 4.0 raM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may excen its action under physiological conditions, depending on the availability of either one of these metal ' ions,

Current Microbiology, 2005

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simu... more Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.

Current Microbiology, 1994

A simple, efficient, and economical method is presented for the preparation of radioactive betain... more A simple, efficient, and economical method is presented for the preparation of radioactive betaine. It involves the incubation of radioactive choline with osmolyte-free Pseudomonas aeruginosa previously grown in hyperosmolar medium with choline as an osmoprotectant. The summarized procedure was as follows: (i) bacteria were grown in high Pi basal salt medium (HPi-BSM) with 20 mM succinate, 18.7 mM NH4C1, 0.8 M NaCl, and 1 mM nonradioactive choline. ,~dter the bacterial pellet was obtained, it was suspended in deionized water to release osmolytes accumulated during growth; (ii) suspension of the pellet, free of osmolytes, in hyperosmolar HPi-BSM with [methyl-14C]-choline (55 nCi/nmol) without the carbon and nitrogen sources. Incubation of the mixture at 37~ for 8-30 h. When only 10% of the initial radioactivity remained in the supernatant, it was withdrawn after centrifugation and the pellet suspended in deionized water. This step released the accumulated betaine plus some contaminants. Purification of betaine contained in the aqueous supernatant was carried out after rotoevaporation to dryness and solubilizatie,n of the residue in methanol. The methanolic extract was rotoevaporated to dryness, the residue solubilized in 10% acetic acid and transferred to a Dowex 50-X8 column. After the column was washed with water and 2 M NHaOH, betaine was eluted by the addition of 4 M NH4OH. The total procedure for obtaining pure radioactive betaine resulted in a yield of 80%. The product obtained was chemically and radiochemically pure, with a specific radioactivity of 54-2-_ 1 nCi/nmol.

… Current Research and …

This paper reports the discovery, plus kinetic, biochemical, biophysical and molecular characteri... more This paper reports the discovery, plus kinetic, biochemical, biophysical and molecular characteristics of a P. aeruginosa phosphorylcholine phosphatase (PchP) whose synthesis depends on choline and derivatives in the culture medium. PchP is the product of the PA5292 gene in the P. ...

Archives of Microbiology, 1990

In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sol... more In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole c a r b o n and nitrogen source, increase acid phosphatase activity. The enzyme activity of both bacteria could be released into the surrounding medium by E D T Alysozyme treatment. The R. meliloti acid phosphatase activity o f crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence o f 5 m M choline, betaine, t r i m e t h y l a m m o n i u m or phosphorylcholine. The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates. A m o n g several phosphoesters tested only pyridoxal-5'-phosphate was hydrolyzed at a considerable rate. In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) o f crude extracts obtained from bacteria grown in the presence o f serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected when alphanaphthylphosphate or pyridoxal-5'-phosphate were used as substrates. In conclusion, although the coline metabolites are capable o f increasing acid phosphatase activities in R. meliloti and in P. aeruginosa, there are two different enzymes involved, apparently in different metabolisms.

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FEMS Microbiology Letters, 1991

Enzyme Research, 2011

Pseudomonas aeruginosaphosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphoryl... more Pseudomonas aeruginosaphosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho), is activated by Mg2+or Zn2+, and is inhibited by high concentrations of substrate. This study has shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and the other in an inhibitory site responsible for the binding of the alkylammonium moiety. The catalytic mechanism for the entry of Pcho in both sites and Zn2+or Mg2+follows a random sequential mechanism. However, Zn2+is more effective than Mg2+at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+induces a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. In contrast, Mg2+produces a relaxed or open conformation.

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium... more Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Bot...

Biology of Reproduction, 1972

Microbiology (Reading, England), 1999

Sterols, carotenoids and gibberellins are synthesized after the reduction of 3-hydroxy-3-methylgl... more Sterols, carotenoids and gibberellins are synthesized after the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate in different subcellular compartments of the fungus Gibberella fujikuroi. Lovastatin inhibits growth in many organisms, presumably because of the inhibition of the synthesis of essential terpenoids. However, in G. fujikuroi growth of the mycelia and sterol and carotenoid content were not affected by the presence of lovastatin. Nevertheless, lovastatin did inhibit the accumulation of gibberellins in the culture medium; this inhibition, however, was counteracted by the addition of mevalonate to the medium. The conversion of HMG-CoA to mevalonate in cell-free extracts was inhibited by 10 nM lovastatin. Since G. fujikuroi apparently possesses a single gene for HMG-CoA reductase, as shown by Southern hybridization and PCR amplification, it was concluded that the biosynthesis of sterols, carotenoids and gibberellins shares a single HMG-CoA reductase, b...

Enzyme Research, 2011

Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, be... more Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP) when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP) or phosphorylcholine (Pcho). The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it ...

Molecular and Cellular Biochemistry, 1990

Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a peripla... more Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg 2+, the K~ values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K~ ~ values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.

Journal of Bacteriology, 2002

The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain... more The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of P. aeruginosa PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in gbt blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.

FEMS Microbiology Letters, 1999

Gibberellins, fatty acids and the polyketides bikaverin and fusarin C are synthesized from a comm... more Gibberellins, fatty acids and the polyketides bikaverin and fusarin C are synthesized from a common precursor, acetyl-CoA. The production of these compounds in Gibberella fujikuroi was strongly influenced by aeration, determined by the air/medium ratio in shaken batch cultures. Higher aeration resulted in increased growth and the production of bikaverin and gibberellins. Low aeration stimulated fatty acid and fusarin C production. Feeding experiments with labeled leucine or acetate resulted in different proportions of labeled palmitic acid and bikaverin, indicating that these compounds are synthesized from independent acetate pools.

FEMS Microbiology Letters, 1998

Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the pr... more Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively. This system obeys Michaelis-Menten kinetics with an apparent K m value of 53 WM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine.

FEMS Microbiology Letters, 1999

Production of bikaverin and gibberellins by Gibberella fujikuroi started after depletion of the n... more Production of bikaverin and gibberellins by Gibberella fujikuroi started after depletion of the nitrogen source, but not after depletion of phosphate. Despite this similarity, the regulation of both pathways by nitrogen involved two different mechanisms. This conclusion was supported by the fact that the production of bikaverin, in contrast to the gibberellins, was not inhibited by nitrate in a mutant that could not utilize it. The different regulation of both pathways was clearly demonstrated by a mutant that overproduced bikaverin but lacked gibberellins. An optimal bikaverin production required a low pH, with a sharp drop at about pH 5. The syntheses of fungal secondary metabolites, such as bikaverin and gibberellins, are not subject to common regulation, but respond to various combinations of signals, such as nitrogen availability, nitrate and the pH of the medium.

FEMS Microbiology Letters, 1989

FEMS Microbiology Letters, 2006

FEBS Letters, 1992

In Pseudomonas aeruginoSao the effect of different cations on the acid phosphatase activity was s... more In Pseudomonas aeruginoSao the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipas¢ C. Although the natural substrate of this enzyme is phosphoryleholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p.nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorycholine phosphatase) mediated by divalent cations Mg 2÷, Zn ~' ÷ and Cu:* revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Ks, values obtained for p-NPP in the presence of Mg "-+, Zn ~' + and Cu:" were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The ICA values for the same ions were 1.25 raM, 0.05 mM and 0.03 raM0 respectively. The Vm,~ obtained in the presence ofCa :+ was about twofold higher than that obtained in the presence of Mg ~ or Zn-'*. The inhibition observed with AI ~" seems to be a multi-site inhibition. The/c',~p and n values, from the Hill plot, were about 0.25 mM and 4.0 raM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may excen its action under physiological conditions, depending on the availability of either one of these metal ' ions,

Current Microbiology, 2005

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simu... more Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.

Current Microbiology, 1994

A simple, efficient, and economical method is presented for the preparation of radioactive betain... more A simple, efficient, and economical method is presented for the preparation of radioactive betaine. It involves the incubation of radioactive choline with osmolyte-free Pseudomonas aeruginosa previously grown in hyperosmolar medium with choline as an osmoprotectant. The summarized procedure was as follows: (i) bacteria were grown in high Pi basal salt medium (HPi-BSM) with 20 mM succinate, 18.7 mM NH4C1, 0.8 M NaCl, and 1 mM nonradioactive choline. ,~dter the bacterial pellet was obtained, it was suspended in deionized water to release osmolytes accumulated during growth; (ii) suspension of the pellet, free of osmolytes, in hyperosmolar HPi-BSM with [methyl-14C]-choline (55 nCi/nmol) without the carbon and nitrogen sources. Incubation of the mixture at 37~ for 8-30 h. When only 10% of the initial radioactivity remained in the supernatant, it was withdrawn after centrifugation and the pellet suspended in deionized water. This step released the accumulated betaine plus some contaminants. Purification of betaine contained in the aqueous supernatant was carried out after rotoevaporation to dryness and solubilizatie,n of the residue in methanol. The methanolic extract was rotoevaporated to dryness, the residue solubilized in 10% acetic acid and transferred to a Dowex 50-X8 column. After the column was washed with water and 2 M NHaOH, betaine was eluted by the addition of 4 M NH4OH. The total procedure for obtaining pure radioactive betaine resulted in a yield of 80%. The product obtained was chemically and radiochemically pure, with a specific radioactivity of 54-2-_ 1 nCi/nmol.

… Current Research and …

This paper reports the discovery, plus kinetic, biochemical, biophysical and molecular characteri... more This paper reports the discovery, plus kinetic, biochemical, biophysical and molecular characteristics of a P. aeruginosa phosphorylcholine phosphatase (PchP) whose synthesis depends on choline and derivatives in the culture medium. PchP is the product of the PA5292 gene in the P. ...

Archives of Microbiology, 1990

In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sol... more In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole c a r b o n and nitrogen source, increase acid phosphatase activity. The enzyme activity of both bacteria could be released into the surrounding medium by E D T Alysozyme treatment. The R. meliloti acid phosphatase activity o f crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence o f 5 m M choline, betaine, t r i m e t h y l a m m o n i u m or phosphorylcholine. The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates. A m o n g several phosphoesters tested only pyridoxal-5'-phosphate was hydrolyzed at a considerable rate. In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) o f crude extracts obtained from bacteria grown in the presence o f serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected when alphanaphthylphosphate or pyridoxal-5'-phosphate were used as substrates. In conclusion, although the coline metabolites are capable o f increasing acid phosphatase activities in R. meliloti and in P. aeruginosa, there are two different enzymes involved, apparently in different metabolisms.

dialnet.unirioja.es

Acceso de usuarios registrados. Acceso de usuarios registrados Usuario Contraseña. ...

FEMS Microbiology Letters, 1991

Enzyme Research, 2011

Pseudomonas aeruginosaphosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphoryl... more Pseudomonas aeruginosaphosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine (Pcho), is activated by Mg2+or Zn2+, and is inhibited by high concentrations of substrate. This study has shown that PchP contains two sites for alkylammonium compounds (AACs): one in the catalytic site near the metal ion-phosphoester pocket, and the other in an inhibitory site responsible for the binding of the alkylammonium moiety. The catalytic mechanism for the entry of Pcho in both sites and Zn2+or Mg2+follows a random sequential mechanism. However, Zn2+is more effective than Mg2+at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+induces a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. In contrast, Mg2+produces a relaxed or open conformation.