Maleylacetate reductase of Pseudomonas sp. strain B13: specificity of substrate conversion and halide elimination

Abstract

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channelling maleylacetate and some chlorinated derivatives into the 3-oxoadipate pathway. Several substituted maleylacetates were prepared in situ by alkaline or enzymatic hydrolysis of dienelactones as the precursor. The conversion of these methyl-, chloro-, fluoro-, and bromo-substituted maleylacetates by maleylacetate reductase from 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. Two moles of NADH per mole of substrate was consumed for the conversion of maleylacetates which contain a halogen substituent in the 2 position. In contrast, only 1 mol of NADH was necessary to convert 1 mol of substrates without a halogen substituent in the 2 position. The conversion of 2-fluoro-, 2-chloro-, 2,3-dichloro-, 2,5-dichloro-, 2,3,5-trichloro-, 2-bromo-, 2,3-dibromo-, 2,5-dibromo-, 2-bromo-5-chloro-, 2-chloro-3-methyl-, and 2-chloro- Widespread use of chlorinated organic compounds presents persistent problems in the environment, particularly because many of these are resistant to biological degradation. A number of these are chlorinated aromatics. Aerobic bacteria use some chloroaromatics as sources of carbon and energy, whereby chlorinated catechols are the central metabolites in the degradation. The chlorocatechol degradation pathway starts with ortho ring cleavage caused by catechol 1,2-dioxygenase giving rise to chloro-cis,cis-muconates (6). The cycloisomerization of the ring cleavage products by chloromuconate cycloisomerase is coupled to chloride elimination leading to (chloro)dienelactones (13). (Chloro)maleylacetates, the compounds resulting from the hydrolysis of the dienelactones by dienelactone hydrolase (13), are further converted to (chloro)-3-oxoadipates by maleylacetate reductase. Thereby, the elimination of chlorine is observed from 2-chloromaleylacetate (8, 17). Maleylacetate reductases have been purified from Pseudomonas sp. strain B13 (9), a 3-chlorobenzoate-degrading strain (5), Alcaligenes eutrophus JMP134 (15), a 2,4-dichlorophenoxyacetic acid-degrading strain (12), and the yeast Trichosporon cutaneum (7) grown with resorcinol.In this article we characterize the substrate specificity of the enzyme from strain B13 with special emphasis on the influence of type and position of substituents (i.e., fluoro, chloro, or bromo) on the elimination of halogen substituents. MATERIALS AND METHODSStrain, culture conditions, and preparation of cell extracts. The media and methods for cultivation of Pseudomonas sp. strain B13 as well as the preparation of cell extracts have been described previously (5, 9).Purification of maleylacetate reductase from strain B13. The maleylacetate reductase of Pseudomonas sp. strain B13 was purified by chromatography on DEAE-cellulose, Butyl-Sepharose, Blue-Sepharose, and Sephacryl S100 as described previously (9). The purity of the enzyme was controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Enzyme assay. Maleylacetate reductase wa...

Metabolism of 5‐Chlorosubstituted Muconolactones

“…Dienelactones have been reported to be converted to maleyl acetate under alkaline conditions as well as by the enzyme dienelactone hydrolase (Evans et al, 1971;Kaschabek and Reineke, 1995). Consequently the formation of 3-methylmaleyl acetate from 3-methyl dienelactones can be expected.…”

“…Detailed analysis of the transformation of substituted maleyl acetates has been reported recently by Kaschabek and Reineke (1995) using maleyl acetate reductase of Pseudomonas sp. B 13.…”

“…Activities were similar to those observed for purified maleyl acetate reductase of Pseudomonas sp. B 13 (Kaschabek and Reineke, 1995).…”

Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway

“…While the enzymes of strains B13 and RHO1 have the same size (they consist of two identical subunits of 37 kDa), the maleylacetate reductase of strain JMP134 was reported to be a dimer composed of two identical subunits of 35 kDa. Besides the function of reducing maleylacetate to give 3-oxoadipate, the enzymes showed dehalogenating activity for maleylacetates with halogen substituents in position 2 so that the convergence of the monochloro-and dichlorocatechol degradation is achieved (13,15,27).…”

Maleylacetate reductases in chloroaromatic-degrading bacteria using the modified ortho pathway: comparison of catalytic properties

“…Interestingly, no specialization has been found for the investigated maleylacetate reductases, although the strains have been enriched from quite different sources with different chloroaromatics: strain B13 was enriched with 3-chlorobenzoate from a k cat values were calculated on the basis of 37,000 and 35,000 for the subunits for strains RHO1 and JMP134, respectively. Kinetic measurements were carried out as described previously (6). The K m values for maleylacetate and 2-chloromaleylacetate correspond to those determined previously with the maleylacetate reductase of strain JMP134 (11).…”