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

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. Ki netic measurement was carried out, in vivo, at 30 degrees C in 50 mM p hosphate buffer (pH 7.0). Under these conditions, the uptake rate was linear for at least 3 min and the value of K-m was 13 mu M. The PATS i s an active transport system that is strongly inhibited by 2,4-dinitro phenol, 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 FATS in starved cells (140%), whereas arse nate (20 mM), NaF, or N,N'-dicyclohexylcarbodiimide (1 mM) did not cau se any effect. Furthermore, the PATS is insensitive to osmotic shock T hese data strongly suggest that the energy for the PATS is derived onl y from an electron transport system which causes an energy-rich membra ne state. The thiol-containing compounds mercaptoethanol, glutathione, and dithiothreitol have no significant effect on the PATS, whereas th iol-modifying reagents such as N-ethylmaleimide and iodoacetate strong ly inhibit uptake (100 and 93%, respectively). Molecular analogs of PA with a substitution (i) on the ring or (ii) on the acetyl moiety or t hose containing (iii) a different ring but keeping the acetyl moiety c onstant inhibit uptake to different extents. None of the compounds tes ted significantly increase the PA uptake rate except adipic acid, whic h greatly stimulates it (163%). The PATS is induced by PA and also, gr atuitously, by some phenyl derivatives containing an even number of ca rbon atoms on the aliphatic moiety (4-phenylbutyric, 6-phenylhexanoic, and 8-phenyloctanoic acids). However, similar compounds with an odd n umber 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. p utida U is cultured in the same medium containing other carbon sources (glucose or octanoic, benzoic, or 4-hydroxyphenylacetic phenylacetic acid) in the place of PA, the PATS and PA-CoA are not detected; neithe r the FATS nor PA-CoA is found in cases in which mutants (PA(-) and PC L(-)) lacking the enzyme which catalyzed the initial step of the PA de gradation (phenylacetyl-CoA ligase) are used. PA-CoA has been extracte d from bacteria and identified as a true PA catabolite by high-perform ance liquid chromatography and also enzymatically with pure acyl-CoA:6 -aminopenicillanic acid acyltransferase from Penicillium chrysogenum.