Anne-Marie Lacoste

Phosphonoacetaldehyde hydrolase from Pseudomonas aeruginosa: purification properties and comparison with Bacillus cereus enzyme.

Phosphonoacetaldehyde hydrolase (2-oxoethylphosphonate phosphonohydrolase, EC 3.11.1.1) has been purified to electrophoretic homogeneity from cells of Pseudomonas aeruginosa A 237 grown in a culture medium containing 2-aminoethylphosphonate as both phosphorus and carbon sources. The native Mr has been estimated to be 62,000 +/- 2000, using a gel filtration column equilibrated with standard proteins. A subunit of Mr 30,000 +/- 1000 determined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gives evidence of a homodimeric structure. The enzyme, which catalyzes the C-P bond cleavage of phosphonoacetaldehyde, has a Km value of 210 microM. It is moderately inhibited by methyl-, ethyl-, propyl- and butylphosphonic acids and activated by aminomethyl-, aminoethylphosphonic acids as well as by phosphonoformic, phosphonoacetic and phosphonopropionic acids. Inhibition by orthophosphite is a time-dependent process which exhibits first-order kinetics and is enhanced by the presence of acetaldehyde. Assays for phosphite removal by dilution or dialysis do not reverse the inhibition. Phosphonoacetaldehyde hydrolase inactivation by phosphite ion appears to be inconsistent with the concept of a Schiff base intermediate as proposed for Bacillus cereus enzyme.

Transport of inorganic phosphate inPseudomonas aeruginosa

Inorganic phosphate transport by wild-typePseudomonas aeruginosa cells grown in a phosphate-limited medium involves a biphasic process. The uptake obeys Michaelis-Menten kinetics with respective apparentKm values of 1.1 μM and 10 μM for the high- and low-affinity systems. These systems may be also differentiated by their sensitivity to osmotic shock, by their specificity towards phosphite, pyrophosphate, arsenate, and some phosphonates and also by their energy requirements. The two phosphate transport systems fromP. aeruginosa are compared with the two major systems (Pst and Pit) characterized inEscherichia coli.

Utilization of 2-aminoethylarsonic acid in Pseudomonas aeruginosa

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (Km 6 microM) was found to be competitively inhibited by AEA and APrA (Ki 18 microM for each). AEP-aminotransferase was found to act on AEA with a Km of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde was generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.

Transamination non enzymatique des acides aminés phosphoniques

Abstract The authors have performed a comparative study of the non-enzymic transamination of ten aminophosphonic acids and their carboxylic analogues with some carbonyl compounds (α-oxo acids, pyridoxal, pyridoxal phosphate). An unfavourable influence of the phosphonic group towards the yield of the transamination reaction is shown by chromatography. On the other hand, a great affinity of α-aminophosphonic acids for pyridoxal phosphate is demonstrated using spectrophotometric methods. These results will emphasize the influence of the acid function, besides other structural factors, in the transamination reaction of amino acids.

Inhibition ofd-alanyl-d-alanine ligase in different bacterial species by amino phosphonic acids

A comparative study was performed on the kinetic properties and the specificity ofd-alanyl-d-alanine ligases fromPseudomonas aeruginosa, Streptococcus faecalis, andStaphylococcus aureus, using some aminophosphonic acids and related compounds.dl-I-Aminoethylphosphonic acid was shown to be a competitive inhibitor of theP. aeruginosa andS. faecalis ligases; assuming ad-form stereospecificity, its activity was nearly equal to that ofd-cycloserine. 2-Aminoethylphosphonate was found to be a weak inhibitor of the ligases, in contrast to the carboxylic analog, β-alanine. γ-Aminobutyric acid and phosphoethanolamine also exhibited some inhibitory properties.

Transport de l'acide amino-2-éthylphosphonique chez Pseudomonas aeruginosa

Summary 2-Aminoethylphosphonic acid (ciliatine) can be used as a source of phosphorus or nitrogen by Pseudomonas aeruginosa. The conditions of its uptake have been investigated. The transport is inducible by ciliatine itself or by its homologue, 3-aminopropylphosphonate, but neither by other phosphonic compounds nor by carboxylic or sulfonic related derivatives. The induction was not suppressed by inorganic phosphate. The transport appears to be an active process, pH and temperature dependent; it requires energy and is dependent on new protein synthesis. The uptake follows Michaelis kinetics. The substrate specificity involved in ciliatine uptake favours the existence of two different transport systems : the first one, inducible by ciliatine, was very sensitive towards different aminophosphonic acids and was competitively inhibited by inorganic phosphate and methylphosphonate; the second transport system, inducible by 3-aminopropylphosphonate, appeared less sensitive towards α-aminophosphonic acids and was non competitively inhibited by phosphate and methylphosphonate. No interactions were observed with related aminocarboxylic acids or with taurine. Some molecular structural requirements for the binding of an effector on both permeases are discussed. The regulatory function of inorganic phosphate, the chief breakdown product of ciliatine, is also emphasized.

Effect of phosphonic analogues of glutamic acid on glutamate decarboxylase

Among the phosphonic analogues of glutamic acid, only 4-amino-4-phosphono butyric acid, the compound which shows the highest affinity for pyridoxal phosphate, inhibits competitively bothEscherichia coli and rat brain glutamate decarboxylases. Phosphinothricin, 2-amino-4-(methylphosphino)butyric acid, is a strong inhibitor of the mammalian enzyme.

In vivo incorporation of cytidine-monophosphate-ciliatine into rat liver lipids.

1. In vivo this investigation was carried out in order to compare the incorporation into rat lipids of free [1,2-minus 14C]-ciliatine and CMP-[1,2-minus 14C]-ciliatine which is the precursor in phosphonolipid biosynthesis. 2. The incorporation of the radioactivity from CMP-[1,2-minus 14C]-ciliatine took place more rapidly than that from free [1,2-minus 14C]-ciliatine in both liver and kidney. The amount of radioactivity from the CMP-[1,2-minus 14C]-ciliatine incorporated into total liver lipids was about 5 times higher than that incorporated into total liver lipids of rat two hrs after injecting free-[1,2-minus 14C]-ciliatine. 3. The amount of [1,2-minus 14C]-ciliatine incorporated into total liver lipids was 15 and 21 times higher than that incorporated into total kidney lipids of rat two and four hrs after injecting free [1,2-minus 14C]-ciliatine. 4. If the main pathway for the phosphonolipid biosynthesis is via CMP-ciliatine, the rate of phosphonolipid formation from CMP-ciliatine must therefore be higher than that from free-ciliatine. The results obtained here indicate therefore that the main pathway for phosphonolipid biosynthesis is a pathway involving CMP-ciliatine. 5. An unknow compound was detected in the water soluble fraction of the acid hydrolyzate of liver phosphonolipids. This material migrated with the N-trimethyl-derivative of ciliatine on the thin-layer chromatogram. The result shows that there is therefore a possibility of methylation of exogenous ciliatine to the phosphonate analogue of choline in the mammalian body.

Aminooxyphosphonates as Slow Binding Inhibitors of Aspartate and Alanine Aminotransferases from Porcine Heart

AbstractAminooxymethylphosphonic (AOMP), 1–aminooxyethylphosphonic (1-AOEP) and 2-aminooxyethyl-phosphonic (2-AOEP) acids have been synthesised and were found to be potent slow binding inhibitors of aspartate- and alanine-aminotransferases with Ki ranging from nanomolar to micromolar values. The half-life of the inhibited complexes varied from 8 min (AspAT-2-AOEP) to 11 h (AspAT-AOMP). Kinetic analysis of the interaction of both enzymes with AOMP suggested the formation of an E-I complex in a single slow binding process. In the case of other compounds, attempt to discriminate between a single- or a double-step mechanism, consistent with an E-I intermediate followed by a slow E-I to E-I* isomerisation process could not be clearly resolved. Spectral studies of the complex formed between PLP-bound enzyme and the aminooxy compound resulted in a shift from 362 nm, the absorption maximum of the native enzyme, to 380 nm, characteristic of the oxime produced. The kinetic parameters for aminooxyphosphonates were c...

Multiplicité des systèmes de transport de la β-alanine et du γ-aminobutyrate chez Pseudomonas aeruginosa

Summary The active transport of β-alanine (β-Ala) and of γ-amino butyric acid (GABA), contrarily to what is observed with α-amino acids, appears to be an inducible process in Pseudomonas aeruginosa A 237 , strain. The transport of β-Ala is induced not only by β-Ala, but also by GABA, β-amino isobutyric acid (β-AIB), 2-amino ethylphosphonic acid (2-AEPh) and 3-amino propylphosphonic acid (3-APrPh). The three above mentioned carboxylic compounds will induce a single β-Ala transport system, whereas their phosphonic analogues give rise to a biphasic uptake. Whatever inducing molecule experimented, β-Ala uptake is inhibited only by GABA and by β-AIB, either competitively (β-Ala or β-AIB induction) or non competitively (GABA, 2-AEPh or 3-APrPh induction). The transport of GABA is similar to that of β-Ala, the same inductors and inhibitors being observed in both cases ; GABA is however the only compound able to induce a single and strictly specific system. The two β-amino acids, β-Ala and β-AIB, inhibit both low and high affinity self-induced systems ; Dixon plots show a purely competitive inhibition at high GABA concentrations and an hyperbolic competitive inhibition at low GABA concentrations. These β-amino acids also appeared as competitive inhibitors for the high affinity systems which were induced by 2-AEPh and 3-APrPh. Despite the fact that β-Ala and β-AIB, as well as 2-AEPh and 3-APrPh, induce transport systems possessing similar kinetics and specificty, the behaviour of the permease activities towards osmotic shock treatment was different : a severe reduction of β-Ala and GABA entry into the cells was observed only after growth on β-Ala, 2-AEPh and GABA. Our results emphasize the complexity of ω-amino acid uptake in Ps. aeruginosa .