p-cymene pathway in Pseudomonas putida: initial reactions

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Abstract
Initial reactions of the p-cymene pathway induced in P. putida PL were reinvestigated. Oxidation of the methyl group attached to the nucleus occurs in 3 steps to give p-cumic acid. The substrate for the ring cleavage of 2,3-dihydroxy-p-cumate is formed from p-cumate in 2 reactions via a dihydrodiol intermediate (2,3-dihydroxy-4-isopropylcyclohexa-4,6-dienoate) and not as previously postulated via 3-hydroxy-p-cumate. There are 3 pieces of evidence for the physiological role of the dihydrodiol intermediate. A mutant of P. putida PL-pT-11/43, which is unable to grow with p-cumate, accumulates a compound from p-cumate, which was identified as 2,3-dihydroxy-4-isoproplycyclohexa-4,6-dienoate. This metabolite is enzymically oxidized by a NAD-dependent dehydrogenase that is present in crude extracts of the wild type and a revertant strain (PL-pT-11/43-R1) but not in the mutant. 3-Hydroxy-p-cumate does not support growth of P. putida PL-W, and it is not oxidized by cells or extracts. 3-Hydroxy-p-cumate was readily isolated as before from culture supernatants, due to its ready formation from the dihydrodiol in acid solution. Mass spectral analysis of the dihydrodiol accumulated in 18O2-enriched atmospheres showed that both hydroxyl atoms are derived from the same molecule of O2. The formation and absorbance maxima of dihydrodiols that accumulate during the growth of the mutant PL-pT-11/43 in the presence of various benzoates (or toluenes) that have substituents at the C-4 atom are reported.

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