Characterization of the last step of the aerobic phenylacetic acid degradation pathway (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.
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.
Bacterial phenylalanine and phenylacetate catabolic pathway revealed
Proceedings of The National Academy of Sciences, 2010
Aromatic compounds constitute the second most abundant class of organic substrates and environmental pollutants, a substantial part of which (e.g., phenylalanine or styrene) is metabolized by bacteria via phenylacetate. Surprisingly, the bacterial catabolism of phenylalanine and phenylacetate remained an unsolved problem. Although a phenylacetate metabolic gene cluster had been identified, the underlying biochemistry remained largely unknown. Here we elucidate the catabolic pathway functioning in 16% of all bacteria whose genome has been sequenced, including Escherichia coli and Pseudomonas putida. This strategy is exceptional in several aspects. Intermediates are processed as CoA thioesters, and the aromatic ring of phenylacetyl-CoA becomes activated to a ring 1,2epoxide by a distinct multicomponent oxygenase. The reactive nonaromatic epoxide is isomerized to a seven-member O-heterocyclic enol ether, an oxepin. This isomerization is followed by hydrolytic ring cleavage and β-oxidation steps, leading to acetyl-CoA and succinyl-CoA. This widespread paradigm differs significantly from the established chemistry of aerobic aromatic catabolism, thus widening our view of how organisms exploit such inert substrates. It provides insight into the natural remediation of man-made environmental contaminants such as styrene. Furthermore, this pathway occurs in various pathogens, where its reactive early intermediates may contribute to virulence. enoyl-CoA hydratase | epoxide | oxepin | oxygenase | phenylacetic acid
Journal of bacteriology, 1994
The phenylacetic acid transport system (PATS) of Pseudomonas putida U was studied after this bacterium was cultured in a chemically defined medium containing phenylacetic acid (PA) as the sole carbon source. Kinetic measurement was carried out, in vivo, at 30 degrees C in 50 mM phosphate buffer (pH 7.0). Under these conditions, the uptake rate was linear for at least 3 min and the value of Km was 13 microM. The PATS is an active transport system that is strongly inhibited by 2,4-dinitrophenol, 4-nitrophenol (100%), KCN (97%), 2-nitrophenol (90%), or NaN3 (80%) added at a 1 mM final concentration (each). Glucose or D-lactate (10 mM each) increases the PATS in starved cells (140%), whereas arsenate (20 mM), NaF, or N,N'-dicyclohexylcarbodiimide (1 mM) did not cause any effect. Furthermore, the PATS is insensitive to osmotic shock. These data strongly suggest that the energy for the PATS is derived only from an electron transport system which causes an energy-rich membrane state. T...
Journal of Biological Chemistry, 2011
The widespread, long sought-after bacterial aerobic phenylalanine/phenylacetate catabolic pathway has recently been elucidated. It proceeds via coenzyme A (CoA) thioesters and involves the epoxidation of the aromatic ring of phenylacetyl-CoA, subsequent isomerization to an uncommon seven-membered C-O-heterocycle (oxepin-CoA), and non-oxygenolytic ring cleavage. Here we characterize the hydrolytic oxepin-CoA ring cleavage catalyzed by the bifunctional fusion protein PaaZ. The enzyme consists of a C-terminal (R)-specific enoyl-CoA hydratase domain (formerly MaoC) that cleaves the ring and produces a highly reactive aldehyde and an N-terminal NADP ؉dependent aldehyde dehydrogenase domain that oxidizes the aldehyde to 3-oxo-5,6-dehydrosuberyl-CoA. In many phenylacetate-utilizing bacteria, the genes for the pathway exist in a cluster that contains an NAD ؉ -dependent aldehyde dehydrogenase in place of PaaZ, whereas the aldehyde-producing hydratase is encoded outside of the cluster. If not oxidized immediately, the reactive aldehyde condenses intramolecularly to a stable cyclic derivative that is largely prevented by PaaZ fusion in vivo. Interestingly, the derivative likely serves as the starting material for the synthesis of antibiotics (e.g. tropodithietic acid) and other tropone/tropolone related compounds as well as for -cycloheptyl fatty acids. Apparently, bacteria made a virtue out of the necessity of disposing the dead-end product with ring hydrolysis as a metabolic branching point. . The abbreviations used are: Paa, phenylacetic acid; Box, benzoate oxidation; ECH, enoyl-CoA hydratase; ALDH, aldehyde dehydrogenase; MBP, maltosebinding protein; RP, reverse phase; LTQ-FT, linear ion trap with Fourier-transform ion cyclotron resonance mass spectrometry; HSQC, heteronuclear single quantum correlation; TOCSY, two-dimensional total correlation spectroscopy; MaoC, monoamine oxidase C.
Archives of Microbiology, 2007
Pseudomonas Xuorescens ST is a styrene degrading microorganism that, by the sequential oxidation of the vinyl side chain, converts styrene to phenylacetic acid. The cluster of styrene upper pathway catabolic genes (sty genes) has been previously localized on a chromosomal region. This report describes the isolation, sequencing and analysis of a new chromosomal fragment deriving from the ST strain genomic bank that contains the styrene lower degradative pathway genes (paa genes), involved in the metabolism of phenylacetic acid. Analysis of the paa gene cluster led to the description of 14 putative genes: a gene encoding a phenylacetyl-CoA ligase (paaF), the enzyme required for the activation of phenylacetic acid; Wve ORFs encoding the subunits of a ring hydroxylation multienzymatic system (paaGHIJK); the gene paaW encoding a membrane protein of unknown function; Wve genes for a -oxidation-like system (paaABCDE), involved in the steps following the aromatic ring cleavage; a gene encoding a putative permease (paaL) and a gene (paaN) probably involved in the aromatic ring cleavage. The function of some of the isolated genes has been proved by means of biotransformation experiments.
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.