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. 1994 Dec;176(24):7667–7676. doi: 10.1128/jb.176.24.7667-7676.1994

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.

C Schleissner 1, E R Olivera 1, M Fernández-Valverde 1, J M Luengo 1
PMCID: PMC197225  PMID: 8002592

Abstract

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. The thiol-containing compounds mercaptoethanol, glutathione, and dithiothreitol have no significant effect on the PATS, whereas thiol-modifying reagents such as N-ethylmaleimide and iodoacetate strongly inhibit uptake (100 and 93%, respectively). Molecular analogs of PA with a substitution (i) on the ring or (ii) on the acetyl moiety or those containing (iii) a different ring but keeping the acetyl moiety constant inhibit uptake to different extents. None of the compounds tested significantly increase the PA uptake rate except adipic acid, which greatly stimulates it (163%). The PATS is induced by PA and also, gratuitously, by some phenyl derivatives containing an even number of carbon atoms on the aliphatic moiety (4-phenyl-butyric, 6-phenylhexanoic, and 8-phenyloctanoic acids). However, similar compounds with an odd number of carbon atoms (benzoic, 3-phenylpropionic, 5-phenylvaleric, 7-phenylheptanoic, and 9-phenylnonanoic acids) as well as many other PA derivatives do not induce the system, suggesting that the true inducer molecule is phenylacetyl-coenzyme A (PA-CoA). Furthermore, after P. putida U is cultured in the same medium containing other carbon sources (glucose or octanoic, benzoic, or 4-hydroxyphenylacetic acid) in the place of PA, the PATS and PA-CoA are not detected; neither the PATS nor PA-CoA is found in cases in which mutants (PA- and PCL-) lacking the enzyme which catalyzed the initial step of the PA degradation (phenylacetyl-CoA ligase) are used. PA-CoA has been extracted from bacteria and identified as a true PA catabolite by high-performance liquid chromatography and also enzymatically with pure acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum.

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Selected References

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  1. Alonso M. J., Bermejo F., Reglero A., Fernández-Cañn J. M., González de Buitrago G., Luengo J. M. Enzymatic synthesis of penicillins. J Antibiot (Tokyo) 1988 Aug;41(8):1074–1084. doi: 10.7164/antibiotics.41.1074. [DOI] [PubMed] [Google Scholar]
  2. Berg D. E., Lodge J., Sasakawa C., Nag D. K., Phadnis S. H., Weston-Hafer K., Carle G. F. Transposon Tn5: specific sequence recognition and conservative transposition. Cold Spring Harb Symp Quant Biol. 1984;49:215–226. doi: 10.1101/sqb.1984.049.01.025. [DOI] [PubMed] [Google Scholar]
  3. Blakley E. R., Halvorson H., Kurz W. The microbial production and some characteristics of delta-carboxymethyl-alpha-hydroxymuconic semialdehyde. Can J Microbiol. 1967 Feb;13(2):159–165. doi: 10.1139/m67-022. [DOI] [PubMed] [Google Scholar]
  4. Blakley E. R., Kurz W., Halvorson H., Simpson F. J. The metabolism of phenylacetic acid by a Pseudomonas. Can J Microbiol. 1967 Feb;13(2):147–157. doi: 10.1139/m67-021. [DOI] [PubMed] [Google Scholar]
  5. DAGLEY S., FEWSTER E., HAPPOLD F. C. The bacterial oxidation of phenylacetic acid. J Bacteriol. 1952 Mar;63(3):327–336. doi: 10.1128/jb.63.3.327-336.1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dagley S. A biochemical approach to some problems of environmental pollution. Essays Biochem. 1975;11:81–138. [PubMed] [Google Scholar]
  7. Evans W. C., Fuchs G. Anaerobic degradation of aromatic compounds. Annu Rev Microbiol. 1988;42:289–317. doi: 10.1146/annurev.mi.42.100188.001445. [DOI] [PubMed] [Google Scholar]
  8. Fernández-Cañn J. M., Reglero A., Martínez-Blanco H., Ferrero M. A., Luengo J. M. Phenylacetic acid transport system in Penicillium chrysogenum Wis 54-1255: molecular specificity of its induction. J Antibiot (Tokyo) 1989 Sep;42(9):1410–1415. doi: 10.7164/antibiotics.42.1410. [DOI] [PubMed] [Google Scholar]
  9. Fernández-Cañn J. M., Reglero A., Martínez-Blanco H., Luengo J. M. Uptake of phenylacetic acid by Penicillium chrysogenum Wis 54-1255: a critical regulatory point in benzylpenicillin biosynthesis. J Antibiot (Tokyo) 1989 Sep;42(9):1398–1409. doi: 10.7164/antibiotics.42.1398. [DOI] [PubMed] [Google Scholar]
  10. Fernández-Valverde M., Reglero A., Martinez-Blanco H., Luengo J. M. Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates. Appl Environ Microbiol. 1993 Apr;59(4):1149–1154. doi: 10.1128/aem.59.4.1149-1154.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Franklin F. C., Lehrbach P. R., Lurz R., Rueckert B., Bagdasarian M., Timmis K. N. Localization and functional analysis of transposon mutations in regulatory genes of the TOL catabolic pathway. J Bacteriol. 1983 May;154(2):676–685. doi: 10.1128/jb.154.2.676-685.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunter D. R., Segel I. H. Acidic and basic amino acid transport systems of Penicillium chrysogenum. Arch Biochem Biophys. 1971 May;144(1):168–183. doi: 10.1016/0003-9861(71)90466-8. [DOI] [PubMed] [Google Scholar]
  14. Luengo J. M., Iriso J. L., López-Nieto M. J. Direct enzymatic synthesis of natural penicillins using phenylacetyl-CoA: 6-APA phenylacetyl transferase of Penicillium chrysogenum: minimal and maximal side chain length requirements. J Antibiot (Tokyo) 1986 Dec;39(12):1754–1759. doi: 10.7164/antibiotics.39.1754. [DOI] [PubMed] [Google Scholar]
  15. Luengo J. M., Iriso J. L., López-Nieto M. J. Direct evaluation of phenylacetyl-CoA: 6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay. J Antibiot (Tokyo) 1986 Nov;39(11):1565–1573. doi: 10.7164/antibiotics.39.1565. [DOI] [PubMed] [Google Scholar]
  16. Luengo J. M., Revilla G., López M. J., Villanueva J. R., Martín J. F. Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum. J Bacteriol. 1980 Dec;144(3):869–876. doi: 10.1128/jb.144.3.869-876.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Martinez-Blanco H., Reglero A., Luengo J. M. Carbon catabolite regulation of phenylacetyl-CoA ligase from Pseudomonas putida. Biochem Biophys Res Commun. 1990 Mar 30;167(3):891–897. doi: 10.1016/0006-291x(90)90607-o. [DOI] [PubMed] [Google Scholar]
  18. Martín-Villacorta J., Reglero A., Ferrero M. A., Luengo J. M. Aliphatic molecules (C-6 to C-8) containing double or triple bonds as potential penicillin side-chain precursors. J Antibiot (Tokyo) 1990 Dec;43(12):1559–1563. doi: 10.7164/antibiotics.43.1559. [DOI] [PubMed] [Google Scholar]
  19. Martín-Villacorta J., Reglero A., Luengo J. M. IV. Acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum: studies on substrate specificity using phenylacetyl-CoA variants. J Antibiot (Tokyo) 1989 Oct;42(10):1502–1505. doi: 10.7164/antibiotics.42.1502. [DOI] [PubMed] [Google Scholar]
  20. Martínez-Blanco H., Reglero A., Fernández-Valverde M., Ferrero M. A., Moreno M. A., Peñalva M. A., Luengo J. M. Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins. J Biol Chem. 1992 Mar 15;267(8):5474–5481. [PubMed] [Google Scholar]
  21. Martínez-Blanco H., Reglero A., Luengo J. M. "In vitro" synthesis of different naturally-occurring, semisynthetic and synthetic penicillins using a new and effective enzymatic coupled system. J Antibiot (Tokyo) 1991 Nov;44(11):1252–1258. doi: 10.7164/antibiotics.44.1252. [DOI] [PubMed] [Google Scholar]
  22. Martínez-Blanco H., Reglero A., Martín-Villacorta J., Luengo J. M. Design of an enzymatic hybrid system: a useful strategy for the biosynthesis of benzylpenicillin in vitro. FEMS Microbiol Lett. 1990 Oct;60(1-2):113–116. doi: 10.1111/j.1574-6968.1990.tb03872.x. [DOI] [PubMed] [Google Scholar]
  23. Martínez-Blanco H., Reglero A., Rodriguez-Aparicio L. B., Luengo J. M. Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid. J Biol Chem. 1990 Apr 25;265(12):7084–7090. [PubMed] [Google Scholar]
  24. Miñambres B., Reglero A., Luengo J. M. Characterization of an inducible transport system for glycerol in Streptomyces clavuligerus. Repression by L-serine. J Antibiot (Tokyo) 1992 Feb;45(2):269–277. doi: 10.7164/antibiotics.45.269. [DOI] [PubMed] [Google Scholar]
  25. Morgan M. S., Darrow R. M., Nafz M. A., Varandani P. T. Participation of cellular thiol/disulphide groups in the uptake, degradation and bioactivity of insulin in primary cultures of rat hepatocytes. Biochem J. 1985 Jan 15;225(2):349–356. doi: 10.1042/bj2250349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
  27. Olivera E. R., Reglero A., Martínez-Blanco H., Fernández-Medarde A., Moreno M. A., Luengo J. M. Catabolism of aromatics in Pseudomonas putida U. Formal evidence that phenylacetic acid and 4-hydroxyphenylacetic acid are catabolized by two unrelated pathways. Eur J Biochem. 1994 Apr 1;221(1):375–381. doi: 10.1111/j.1432-1033.1994.tb18749.x. [DOI] [PubMed] [Google Scholar]
  28. Rodríguez-Aparicio L. B., Reglero A., Luengo J. M. Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system. Biochem J. 1987 Sep 1;246(2):287–294. doi: 10.1042/bj2460287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Selvaraj G., Iyer V. N. Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. J Bacteriol. 1983 Dec;156(3):1292–1300. doi: 10.1128/jb.156.3.1292-1300.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vitovski S. Phenylacetate-coenzyme A ligase is induced during growth on phenylacetic acid in different bacteria of several genera. FEMS Microbiol Lett. 1993 Mar 15;108(1):1–5. doi: 10.1016/0378-1097(93)90477-j. [DOI] [PubMed] [Google Scholar]
  31. Wheelis M. L., Stanier R. Y. The genetic control of dissimilatory pathways in Pseudomonas putida. Genetics. 1970 Oct;66(2):245–266. doi: 10.1093/genetics/66.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]

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