Identification and Purification of Distinct Isomerase and Decarboxylase Enzymes Involved in the 4‐Hydroxyphenylacetate Catabolic Pathway of Escherichia coli

Abstract

The possible involvement of an isomerase in the 4-hydroxyphenylacetate meta-cleavage pathway has been studied. 5-Carboxymethyl-2-hydroxymuconate has been shown to undergo both spontaneous and enzymecatalysed isomerisation to give 5-carboxymethyl-2-oxo-hex-3-ene-1,6-dioate, a compound with an absorbance maximum at 246 nm. The latter compound rather than the former is the substrate for a decarboxylase that produces 2-hydroxyhepta-2,4-diene-1,7-dioate. The isomerase and decarboxylase enzymes have been purified to over 90 % homogeneity. Mg2+ is required for the decarboxylase reaction but not for the isomerase.In an investigation of the meta-cleavage reaction sequence for the catabolism of 4-hydroxyphenylacetate by Pseudomonas putida it was suggested that an enzyme-catalysed isomerisation of 5-carboxymethyl-2-hydroxymuconate might occur prior to its decarboxylation [l]. Such an isomerisation would be analogous to that occurring for 2-hydroxymuconate in the metacleavage degradation of catechol by Azotobacter and Pseudomonas species [2,3].Until very recently there was no published experimental evidence in support of this proposal but now Barbour and Bayly [4] have described experiments with Pseudomonas mutants, which they interpret as evidence for distinct isomerase and decarboxylase enzymes involved in 4-hydroxyphenylacetate catabolism.Escherichia coli degrades 4-hydroxyphenylacetate by the same meta-cleavage pathway [S] and we have been investigating the problem by purification of the decarboxylase activity from E. coli. In the process we found that distinct isomerase and decarboxylase enzymes could be obtained.The purified enzymes have been used to study the isomerase and decarboxylase substrates and we find that the spectral characteristics of these compounds are different from those ascribed to them from the use of mutant extracts [4]. MATERIALS AND METHODS Bacteria and Culture MethodsThe organism used was Escherichia coli strain B. Cells were cultured on 4-hydroxyphenylacetate as described previously [6]. Preparation of ExtractsExtracts were prepared by ultrasonication of cells suspended in 0.09 M Na2HP04/KH2P04 buffer pH 7.0, con- S.3.3.-); S-carboxyrnethyl-2-oxo-hex-3-ene-l,h-dioate decarboxylase (EC 4.1.1. ). Enzymes. 5-Carboxymethyl-2-hydroxymuconate isomerase (ECtaining 9 % glycerol and 1 mM dithiothreitol, as described previously [6]. Protein EstimationProtein was measured either colorimetrically, with crystalline bovine serum albumen as standard, by the modified Folin method [7] or spectrophotometrically [8].Enzyme Assays 5-Carboxymethyl-2-hydroxymuconate isomerase activity was assayed by measuring the decrease in absorption at 300 nm consequent upon the formation of 5-carboxymethyl-2-oxo-hex-3-ene-1,6-dioate. The reaction mixture contained 0.045 pmol 5-carboxymethyl-2-hydroxymuconate in 1 .0 ml of 0.1 M Na2HP04/KH2P04 buffer pH 7.5 at 30°C. The rate of spontaneous isomerisation was measured for 60 s after addition of the substrate and the reaction was then initiated by addition of enzyme. The initial rat...

Genomic and proteomic analysis of lignin degrading and polyhydroxyalkanoate accumulating β-proteobacterium Pandoraea sp. ISTKB

“…The analysis of expression profile on KL indicates the presence of metacleavage and unusual pathways, i.e., ‘-CoA’-mediated degradation of lignin derivatives in aerobic microorganisms. The presence of 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase in the expression profile of KL possibly indicated 4-hydroxyphenylacetate degradation through meta cleavage pathways [ 28 ]. Benzoyl-CoA oxygenase-mediated degradation of aromatic compound is completely different mechanisms and observed in 4–5% of sequenced bacterial genomes.…”

Molecular Adaptation Mechanisms Employed by Ethanologenic Bacteria in Response to Lignocellulose-derived Inhibitory Compounds

“…The degradation of these inhibitory compounds has been widely demonstrated in many bacterial species, particularly for phenolics, where meta/ortho-cleavage and β-ketoadipate pathways are used for phenolic compound degradation 125-128. Phenolic degradation pathways have been previously described in Gram positive bacteria genera, such as Aerobacter 129 Arthrobacter 130, Bacillus 131-135, Lactobacillus 136, 137, Rhodococcus 138 and Paenibacillus 139, as well as in Gram negative bacteria genera such as Acinetobacter 140, Comamonas 141, 142, Enterobacter 143, Escherichia 144-148, Klebsiella 149, Pseudomonas 150-153 and Sphingomonas 154, 155. It has been shown that these microorganisms have evolved with abilities to degrade phenolics such as ferulic acid, vanillic acid, protocatechuic acid, and catechol, and be able to break them down into simple products via series of enzymatic processes, which can then channelled into the TCA and/or glyoxylate cycle(s) to produce energy (Figure 5) 125-128, 131, 139, 147, 150.…”

4-Oxalocrotonate Tautomerase, Its Homologue YwhB, and Active Vinylpyruvate Hydratase: Synthesis and Evaluation of 2-Fluoro Substrate Analogues

“…YcgM is a putative isomerase found in E. coli. COHED is one of the enzymes in the homoprotocatechuate meta-fission pathway, an inducible set of enzymes in E. coli C that converts 3,4-dihydroxyphenylacetate (30, Scheme 6) to succinic semialdehyde and pyruvate (27). The enzymes of this pathway are part of a degradative route for phenylalanine and tyrosine, and they carry out reactions analogous to those in the catechol meta-fission pathway on substrates in which a carboxymethyl group has replaced the C-5 hydrogen of 1.…”

“…The activity of 4-OT was determined by a previously described assay using 1 (4), following the increase in absorbance at 236 nm due to the formation of 5. The activity of YwhB was determined by a previously described assay using phenylpyruvate (27). Ketonization of 27 to generate 28 is followed by the decay at 288 nm (22).…”

“…The FAH superfamily consists of three structurally characterized members: YcgM; 5-(carboxymethyl)-2-oxo-3hexene-1,6-dioate decarboxylase (COHED), which catalyzes the decarboxylation of a structurally similar substrate to that of 5 (27); and the mouse enzyme, fumarylacetoacetate hydrolase (FAH) (28). In the crystal structure of a COHED‚ calcium complex, three residues, Glu-276, Glu-278, and Asp-307, are coordinated to the metal ion (29).…”