Oxidative metabolism of protocatechuic acid by certain soil pseudomonads: a new ring-fission mechanism

Abstract

Pathways of phthalate metabolism by Pseudomonas P 1 and Pseudomonas D2 and of p-cresol by Pseudomonas D 1 converge at the protocatechuate stage. Extracts of Pseudomonas P 1 oxidized proto-catechuate via cis-cis-[beta]-carboxymuconate to [beta]-oxoadipate with the consumption of 1 mole of CO2 and the evolution of 1 mole of CO2/mole of substrate. Protocatechuate is oxidized by cell-free extracts of Pseudomonas D 1 and of Pseudomonas D 2 with the consumption of 1 mole of 02/mole of substrate, but no CO2 is evolved. The product, gamma-carboxy-a-hydroxymuconic semialdehyde, results from an oxidative cleavage at the 4,5-bond; the new ring-fission enzyme has been called protocatechuate 4,5-oxygenase. The structure assigned to the new ring-fission product, gamma-carboxy-[alpha]-hydroxymuconic semialdehyde, is based on the following evidence: production of 1 new carboxyl group/molecule during ring fission; analysis of a crystalline acidic bis-2,4-dinitrophenylhydrazone derivative; nature of the absorption spectrum curve in acid and alkali suggesting tautomeric change of keto and enol forms; production of 2,4-lutidinic acid by the action of NH4+ ions. A scheme for the further metabolism of gamma-carboxy-[alpha]-hydroxy-muconic semialdehyde to pyruvate and malate via 4-carboxy-2,3-dihydro-2-hydroxymuconate is proposed. The pH optimum of protocatechuate 4,5-oxygenase is 7-0-7-5; Fe2+ ions and a reducing compound are essential for full activity.