Characterization of muconate and chloromuconate cycloisomerase from Rhodococcus erythropolis 1CP: indications for functionally convergent evolution among bacterial cycloisomerases

Abstract

Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were purified from extracts of Rhodococcus erythropolis 1CP cells grown with benzoate or 4-chlorophenol, respectively. Both enzymes discriminated between the two possible directions of 2-chloro-cis,cis-muconate cycloisomerization and converted this substrate to 5-chloromuconolactone as the only product. In contrast to chloromuconate cycloisomerases of gram-negative bacteria, the corresponding R. erythropolis enzyme is unable to catalyze elimination of chloride from (؉)-5-chloromuconolactone. Moreover, in being unable to convert (؉)-2-chloromuconolactone, the two cycloisomerases of R. erythropolis 1CP differ significantly from the known muconate and chloromuconate cycloisomerases of gram-negative strains. The catalytic properties indicate that efficient cycloisomerization of 3-chloro-and 2,4-dichloro-cis,cis-muconate might have evolved independently among gram-positive and gram-negative bacteria.Many chloroaromatic compounds are degraded by bacteria via chlorocatechols as central intermediates. Further catabolism involves ortho-cleavage of the chlorocatechols to chlorosubstituted cis,cis-muconates as well as cycloisomerization and dechlorination of the latter, yielding dienelactones (4-carboxymethylenebut-2-en-4-olides) which are hydrolyzed and finally funneled into the ubiquitous 3-oxoadipate pathway (Fig. 1). Despite much of the early work having been done with an Arthrobacter sp. (3,8,31) and despite many reports of transformation of halogenated aromatic compounds by gram-positive bacteria (recently reviewed in reference 35), the enzymology and genetics of the modified ortho-cleavage pathway outlined above have been elucidated almost exclusively in gram-negative strains. They usually contain separate sets of enzymes for catechol and chlorocatechol conversion, which differ from each other with respect to the affinities and turnover rates for chlorosubstituted catechols or the metabolites formed from them (6,21,25).The gram-positive strain Rhodococcus erythropolis 1CP has previously been reported to utilize 4-chlorophenol and 2,4-dichlorophenol as sole sources of carbon and energy (10). After some adaptation, it also grows slowly with 3-chlorophenol but not with 2-chlorophenol. Like many gram-negative strains, R. erythropolis 1CP possesses separate catechol and chlorocatechol catabolic enzymes (14, 16). The substrate preferences of the chlorocatechol 1,2-dioxygenase (15) and of the dienelactone hydrolase (16) of R. erythropolis 1CP suggest that, corresponding to the growth substrates, only a 4-chlorocatechol branch and a 3,5-dichlorocatechol branch are functional in strain 1CP, but there is no 3-chlorocatechol branch (Fig. 1). In this paper, we show that the substrate preference of the R. erythropolis 1CP chloromuconate cycloisomerase fits well with those of the dioxygenase and of the hydrolase. Moreover, 2-chloro-cis,cis-muconate was found to be converted to only one product, 5-chloromuconolactone, by both the muconate and the ...

Metabolism of 5‐Chlorosubstituted Muconolactones

“…Whereas bacterial cycloisomerases catalyze 3,6-~ycloisomerization of 3-chloro-and 3-methyl muconate (Catelani et al, 1971 ;Knackmuss et al, 1976;, fungal enzymes catalyze 1,4-~ycloisomerization (Cain et al, 1989b;Mazur et al, 1994). Differences in cycloisomerization were also observed with 2-chloro-cis,cis-muconate even among bacterial enzymes, but more importantly, the property to catalyze dehalogenation is, to our current knowledge, restricted to chloromuconate cycloisomerases of Gram-negative microorganisms Solyanikova et al, 1995).…”

Formation of Protoanemonin from 2-Chloro-cis,cis-Muconate by the Combined Action of Muconate Cycloisomerase and Muconolactone Isomerase

“…Most muconate cycloisomerases described thus far catalyze a 1,4-cycloisomerization to form 2CML (reaction A) and a 1,6-cycloisomerization to form 5-CML (reaction B) (29). Muconate cycloisomerase isolated from Rhodococcus erythropolis 1CP catalyze reaction B only (27). Chloride elimination from 5CML to give transdienelactone is catalyzed exclusively by chloromuconate cycloisomerases (28).…”

“…Also, 2CML, in contradiction to previous assumptions, was biologically active and was converted to trans-dienelactone via 2-chloromuconate and 5CML by chloromuconate cycloisomerase. In addition to those enzymes purified from gram-negative bacteria, muconate cycloisomerases with distinct catabolic properties have been purified from Rhodococcus strains (27). None of the Rhodococcus enzymes could dehalogenate 2-chloromuconate, and 5CML was the only reaction product, showing that the enzymes discriminate between the different cycloisomerization possibilities.…”

Crystal Structure of 4-Chlorocatechol 1,2-Dioxygenase from the Chlorophenol-utilizing Gram-positive Rhodococcus opacus 1CP

“…Many of these enzymes have been characterized extensively in terms of biochemical, spectroscopic, and kinetic properties (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Mechanistic and structural details have also been gathered for one 1,2-CTD (42) and several 3,4-PCDs (43)(44)(45)(46)(47).…”