Inability of muconate cycloisomerases to cause dehalogenation during conversion of 2-chloro-cis,cis-muconate

Abstract

The conversion of 2-chloro-cis,cis-muconate by muconate cycloisomerase from Pseudomonas putida PRS2000 yielded two products which by nuclear magnetic resonance spectroscopy were identified as 2-chloro-and 5-chloromuconolactone. High-pressure liquid chromatography analyses showed the same compounds to be formed also by muconate cycloisomerases from Acinetobacter calcoaceticus ADP1 and Pseudomonas sp. strain B13. During 2-chloro-cis,cis-muconate turnover by the enzyme from P. putida, 2-chloromuconolactone initially was the major product. After prolonged incubation, however, 5-chloromuconolactone dominated in the resulting equilibrium. In contrast to previous assumptions, both chloromuconolactones were found to be stable at physiological pH. Since the chloromuconate cycloisomerases of Pseudomonas sp. strain B13 and Akaligenes eutrophus JMP134 have been shown previously to produce the trans-dienelactone (trans4-carboxymethylenebut-2-en-4-olide) from 2-chloro-cis,cis-muconate, they must have evolved the capability to cleave the carbonchlorine bond during their divergence from normal muconate cycloisomerases.The bacterial mineralization of chloroaromatic compounds necessarily involves the cleavage of carbon-chlorine bonds, liberating inorganic chloride. In several cases, this is achieved prior to the opening of the aromatic ring by reductive, oxygenolytic, or hydrolytic reactions (for reviews, see references 12, 19, and 45). In many other cases, however, chloride elimination occurs only after ring cleavage has been accomplished. Corresponding catabolic pathways have been described mainly for mono-and dichlorosubstituted phenoxyacetates, phenols, benzoates, benzenes, anilines, salicylates, and metabolic precursors of them. Under aerobic conditions, all of these compounds are usually first converted to chlorocatechols as central intermediates. These are then subject to intradiol (ortho) ring cleavage, giving rise to chlorosubstituted cis,cismuconates. It has long been known (3,13,14,57) that chloride elimination on this pathway is related to the conversion of mono-and dichloromuconates to unsubstituted or chlorosubstituted dienelactones (4-carboxymethylenebut-2-en-4-olides) (Fig. 1). The dienelactones are then cleaved hydrolytically, and the products are finally funneled into the 3-oxoadipate pathway (10,13,14,24,48,52,57,60).Bacteria, employing the modified ortho cleavage pathway outlined above, usually do so by inducing a set of plasmidencoded enzymes which are specially adapted for the turnover of chlorocatechols or their respective metabolites (9,41,48). With the exception of maleylacetate reductase, these enzymes catalyze reactions analogous to those of the ordinary 3-oxoadipate pathway.Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were first differentiated by Schmidt and Knackmuss (48) in the 3-chlorobenzoate-utilizing strain Pseudomonas sp. strain B13. The authors reported that both enzymes catalyze basically the same reactions and that they differ only with respect to their...

Metabolism of 5‐Chlorosubstituted Muconolactones

“…In the preceding paper it is shown that muconolactone isomerase is able to transform 5-chlorosubstituted muconolactones (Prucha et al, 1996). We report here on Vollmer et al (1994). Enzymes involved are: 1, muconate cycloisomerase; 2, chloromuconate cyclisomerase; 3, muconolactone isomerase; 4, 3-oxoadipate enol-lactone hydrolase; 5, 4-methylmuconolactone methyl-isomerase; 6, muconolactone isomerase or isoenzyme modified for the metabolism of methyl substituted analogues; 7, 3-oxoadipate enol-lactone hydrolase or isoenzyme modified for the metabolism of methyl substituted analogues ; 8, dienelactone hydrolase ; 9, maleyl acetate reductase; 10, acid catalyzed reaction.…”

“…Attempts to clarify the configuration by transformation into a rigid bromodilactone, as described by Cain et al (1989a) for the determination of the configuration of (4s)-( -)-3-methylmuconolactone, failed due to the high instability of 5-chloro-3-methylmuconolactone even under slightly alkaline conditions (data not shown). A definite assignment (Table 8) can now be made based on the 'H-NMR data (Pieper et al, 1993), the results of chemical transformation experiments and the comparison with the fate of biologically produced 5-chloromuconolactone (Vollmer et al, 1994) which has the (4R, SS)-configuration. It has to be stated here that a (4R)-configuration of 5-chlorosubstituted muconolactones corresponds to a (4s)-configuration in the previously described 3-methyl-, 4-methyl-and unsubstituted muconolactones (Cain et al, 1989a;Chari et al, 1987).…”

“…Transformation of 5-chloromuconolactone. 5-Chloromuconolactone was reported by Vollmer et al (1994) to be one product formed by transformation of 2-chloro-cis,cis-muconate by muconate cycloisomerases (Fig. 1 C).…”

“…5-Chloromuconolactone was prepared by a variation of the method of Vollmer et al (1994). The reaction mixture contained the following components : muconate cycloisomerase from Alcaligenes eutrophus JMP 134, partially purified by anion-exchange chromatography as described by Kuhm et al (1990) and with a total activity of 30 U (as measured with &,cis-muconate at pH S), in 30 mM BistrisMCI, 1 mM MnCl,, 2 mM 2-chlorocis,cis-muconate.…”

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

“…Chloromuconate cycloisomerases catalyze a dehalogenation of 3-chloromuconate to form cis-dienelactone (53), and muconate cycloisomerases catalyze the formation of protoanemonin (1) with 4-chloromuconolactone as a reaction intermediate (1,32). 2-Chloromuconate is dehalogenated only by chloromuconate cycloisomerases (59), while mu-conate cycloisomerases form 2-chloro-and 5-chloromuconolactone (60).…”