Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad. Ring-fission, lactonizing and delactonizing enzymes

Abstract

1. A cell-free system, prepared from Pseudomonas N.C.I.B. 9340 grown on 4-chloro-2-methylphenoxyacetate (MCPA) was shown to catalyse the reaction sequence: 5-chloro-3-methylcatechol → cis–cis-γ-chloro-α-methylmuconate → γ-carboxymethylene-α-methyl-Δαβ-butenolide → γ-hydroxy-α-methylmuconate. 2. The activity of the three enzymes involved in these reactions was completely resolved and the lactonizing and delactonizing enzymes were separated. 3. This part of the metabolic pathway of 4-chloro-2-methylphenoxyacetate is thus confirmed for this bacterium. 4. The ring-fission oxygenase required Fe2+ or Fe3+ and reduced glutathione for activity; the lactonizing enzyme is stimulated by Mn2+, Mg2+, Co2+ and Fe2+; no cofactor requirement could be demonstrated for the delactonizing enzyme. 5. cis–cis-γ-Chloro-α-methylmuconic acid was isolated and found to be somewhat unstable, readily lactonizing to γ-carboxymethylene-α-methyl-Δαβ-butenolide. 6. Enzymically the lactonization appears to be a single-step dehydrochlorinase reaction.