Aerobic catabolism of phenylacetic acid in Pseudomonas putida U: biochemical characterization of a specific phenylacetic acid transport system and formal demonstration that phenylacetyl-coenzyme A is a catabolic intermediate (original) (raw)
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Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).
European Journal of Biochemistry, 1994
Phenylacetic acid (PhAcOH) and 4-hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonus putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups : those blocked in the gene encoding 4-hydroxyphenylacetic acid-3-hydroxylase ; and those blocked in the gene encoding homoprotocatechuate-2,3-dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups : those which contain a functional PhAc-CoA ligase protein; and those lacking this enzyme activity.
Characterization of the last step of the aerobic phenylacetic acid degradation pathway
Microbiology, 2007
Phenylacetic acid (PA) degradation in bacteria involves an aerobic hybrid pathway encoded by the paa gene cluster. It is shown here that succinyl-CoA is one of the final products of this pathway in Pseudomonas putida and Escherichia coli. Moreover, in vivo and in vitro studies revealed that the paaE gene encodes the b-ketoadipyl-CoA thiolase that catalyses the last step of the PA catabolic pathway, i.e. the thiolytic cleavage of b-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA. Succinyl-CoA is suggested as a common final product of aerobic hybrid pathways devoted to the catabolism of aromatic compounds.
Gene, 2004
Pseudomonas sp. strain Y2 is a styrene degrading bacterium that mineralises this compound through its oxidation to phenylacetic acid (PAA). We previously identified a complete gene cluster (paa1 cluster) for the degradation of phenylacetate, but, surprisingly, some paa1 deletion mutants were still able to catabolize styrene (STY) suggesting that this strain contained a second catabolic pathway. We report here the characterization of a second and novel paa2 gene cluster comprising 17 genes related to the catabolism of phenylacetate. We have identified a new gene (paaP) that is most likely involved in a transport process. Remarkably, the organization of the paa2 gene cluster is more similar to that of Pseudomonas putida KT2440 than to the paa1 gene cluster. Two new genes of undefined function were located inside the paa2 cluster. Sequence comparison between the paa2 genes and the paa1 and paa clusters of Pseudomonas sp. strain Y2 and P. putida KT2440, respectively, revealed a similar degree of divergence among the three sets of genes. Differences in the gene organization between paa1 and paa2 clusters of Pseudomonas sp. strain Y2 can be explained by an independent evolutionary history, probably associated with the adjacent sty genes. Deletion of either the first (paa1) or the second (paa2) gene cluster did not affect the ability of strain Y2 to grow in phenylacetate, whereas the deletion of both clusters led to the loss of this ability. The co-existence of two functional gene clusters for the degradation of phenylacetic acid in a bacterium has not been reported so far. D Abbreviations: bp, base pair(s); kb, 1000 bp; aa, amino acid(s); sty, gene(s) encoding protein(s) involved in the oxidation of styrene to phenylacetic acid; paa, gene(s) encoding protein(s) involved in the catabolism of phenylacetic acid; PAA, phenylacetic acid.
Archives of Microbiology, 1993
The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate + CoA + ATP-+ phenylacetyl-CoA + AMP § PPi and requires Mg 2+. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 gmol minx rag-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 ~tmol min-1 rag-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 + 2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn 2 + can substitute for Mg 2 +. The apparent K m values for phenylacetate, CoA, and ATP are 60, 150, and 290 gM, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzyme was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.
FEMS Microbiology Letters, 2006
Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and benzoate as the sole source of carbon and energy. Compared with glucose, cells grew faster on aromatic compounds as well as on organic acids. The organism failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. Whole-cell oxygen uptake, enzyme activity and metabolic studies suggest that in strain CSV86 glucose utilization is exclusively by the intracellular phosphorylative pathway, while in Stenotrophomonas maltophilia CSV89 and P. putida KT2442 glucose is metabolized by both direct oxidative and indirect phosphorylative pathways. Cells grown on glucose showed five-to sixfold higher activity of glucose-6-phosphate dehydrogenase compared with cells grown on aromatic compounds or organic acids as the carbon source. Study of [ 14 C]glucose uptake by whole cells indicates that the glucose is taken up by active transport. Metabolic and transport studies clearly demonstrate that glucose metabolism is suppressed when strain CSV86 is grown on aromatic compounds or organic acids.
The Journal of Antibiotics, 1989
The phenylacetic acid (PA) transport system of Penitillium chrysogenum is induced by PA, 2-hydroxyphenylacetic and 4-phenylbutyric acids but not by benzoic, phenoxyacetic acid and phenylpropionic acids. Substitution in the aromatic moiety (3-hydroxyphenylacetic, 4-hydroxyphenylacetic acids), replacement of the aromatic moiety by other rings (thiophene-2-acetic acid, indole-3-acetic or indole-3-butyric acids) or the presence of an amino group in the a-position (2-aminophenylacetic acid) eliminates inducing activity. 2-Phenylbutyric acid dose not induce the PA transport system indicating that fatty acid-/S-oxidation is needed to generate the authentic regulatory molecule (phenylacetyl-CoA) from 4-phenylbutyric acid. Furthermore, the uptake system synthesized in presence of PA, 2-hydroxyphenylacetic or 4-phenylbutyric acids is under carbon catabolic repression control and is also repressed by L-lysine suggesting that the three molecules induce in P. chrysogenuma single mechanismof transport.
The Journal of Antibiotics, 1989
The phenylacetic acid (PA) transport system in Penicillium chrysogenum is an induciblesystem (see Fernandez-Canon et al ; preceding papers) which is repressed by free amino acids when these molecules are added to the complex fermentation broths at the induction time. L-Tyrosine, L-«:-aminoadipic acid, L-tryptophan, L-phenylalanine and L-methionine are the molecules that cause the greatest delay in induction. The addition of Krebs-cycle intermediates to the complex fermentation broth did not affect the rate of induction with the exception of oxalacetic acid and citric acid which strongly increased it. Ammonium salts and acetate also repressed the biosynthesis of the enzymes involved in the PAuptake.