title: Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida.
abstract: The meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds. In this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route. In the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the actions of 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxovalerate aldolase. In this study, the isomerase, decarboxylase, and hydratase encoded in the TOL plasmid pWW0 of Pseudomonas putida mt-2 were purified and characterized. The 28-kilodalton isomerase was formed by association of extremely small identical protein subunits with an apparent molecular weight of 3,500. The decarboxylase and the hydratase were 27- and 28-kilodalton polypeptides, respectively, and were copurified by high-performance-liquid chromatography with anion-exchange, hydrophobic interaction, and gel filtration columns. The structural genes for the decarboxylase (xylI) and the hydratase (xylJ) were cloned into Escherichia coli. The elution profile in anion-exchange chromatography of the decarboxylase and the hydratase isolated from E. coli XylI+XylJ- and XylI-XylJ+ clones, respectively, were different from those isolated from XylI+ XylJ+ bacteria. This suggests that the carboxylase and the hydratase form a complex in vivo. The keto but not the enol form of 4-oxalocrotonate was a substrate for the decarboxylase. The product of decarboxylation was 2-hydroxypent-2,4-dienoate rather than its keto form, 2-oxopent-4-enoate. The hydratase acts on the former but not the latter isomer. Because 2-hydroxypent-2,4-dienoate is chemically unstable, formation of a complex between the decarboxylase and the hydratase may assure efficient transformation of this unstable intermediate in vivo.
authors.affiliation: Department of Medical Biochemistry, University of Geneva, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Harayama
authors.forename: M
authors.initials: M
authors.lastname: Rekik
authors.forename: K L
authors.initials: KL
authors.lastname: Ngai
authors.forename: L N
authors.initials: LN
authors.lastname: Ornston
mesh_terms: D002262:Carboxy-Lyases / Q000378:metabolism; D002396:Catechols / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002396:Catechols; D005231:Fatty Acids, Unsaturated; C061660:2-hydroxy-2,4-pentadienoic acid; C021917:4-oxalocrotonate decarboxylase; D002262:Carboxy-Lyases; C021417:4-hydroxy-2-oxovalerate aldolase; D007652:Oxo-Acid-Lyases; D006836:Hydro-Lyases; C061677:2-oxopent-4-enoate hydratase; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases; C034221:catechol