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

Abstract

The maleylacetate reductases from Pseudomonas aeruginosa RHO1 and Alcaligenes eutrophus JMP134 were tested for activity and affinity to various maleylacetates as well as dechlorinating properties. The dechlorinating activity and the k cat /K m values revealed high-level similarity of these reductases to that of Pseudomonas sp. strain B13.Various chloroaromatics are degraded by bacteria via chlorocatechols as central intermediates. After several steps in the following modified ortho cleavage pathway, (chloro)maleylacetate is formed. The degradation of 2,4,6-trichloro-and pentachlorophenol as well as 2,4,5-trichlorophenoxyacetate, which involves (chlorinated) 1,2,4-trihydroxybenzene as the central intermediate, also gives rise to (chloro)maleylacetate.In general, (chloro)maleylacetate is further converted to (chlorinated) 3-oxoadipate by a maleylacetate reductase (Fig. 1). In the present paper we compare maleylacetate reductases of bacteria, enriched with different chloroaromatic compounds, with respect to their substrate specificities.Maleylacetate reductase was purified to homogeneity from 1,4-dichlorobenzene-grown cells of Pseudomonas aeruginosa RHO1, according to the method of Kaschabek and Reineke (5). During the purification the specific activity of the enzyme increased to 170 U/mg of protein, indicating a 74-fold purification with 13% recovery of activity. The M r of the native protein was determined by gel filtration on Superose 6 to be approximately 74,000. M r estimation of the subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single band with a value of 37,000. The isoelectric point of the purified enzyme was determined by the method of O'Farrell to be 7.0. The optimum activity was determined to be at pH 5.8 in 50 mM histidine-HCl buffer. Activity was drastically reduced at pH values below 5.8. Examination of the temperature influence showed that in 50 mM Tris-HCl buffer, pH 7.0, the highest level of enzyme activity could be observed at 45ЊC. Furthermore, inhibitory experiments with chelating agents, cyanide ions, and p-chloromercuribenzoate showed that the maleylacetate reductase of strain RHO1 strongly resembles that of Pseudomonas sp. strain B13 (5).In general, the enzymes of strain RHO1 and Alcaligenes eutrophus JMP134 showed broad substrate specificities (Table 1). Both reductases required 2 mol of NADH per molequivalent of 2-halogenated maleylacetates for the complete turnover of these substrates. In contrast, only 1 mol of NADH was consumed with substrates bearing no halogen substituent in position 2. The reduction of the 2-halogenated maleylacetates resulted in the elimination of 1 molequivalent of the corresponding halide, while halomaleylacetates missing a halogen substituent in position 2 showed no elimination of halide. These properties are shared with the enzyme of strain B13 (6). Overall, both enzymes revealed low-level activities with methylmaleylacetates. 3-Bromomaleylacetate was found to be the best substrate for maleylacetate reductases.The enzymology of...

Genuine genetic redundancy in maleylacetate-reductase-encoding genes involved in degradation of haloaromatic compounds by Cupriavidus necator JMP134

“…The two plasmid-encoded MAR in C. necator JMP134 have been extensively studied (Kasberg et al, 1995;Muller et al, 1996;Seibert et al, 1993;Vollmer et al, 1993). Both pJP4-encoded MAR genes (tfdF I and tfdF II ) are functional and contribute to catabolism of chlorocatechols (Plumeier et al, 2002).…”

Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation byPseudomonas reinekeiMT1

“…The ccaD gene obviously encodes a MAR. Genes encoding MARs have initially been observed in chlorocatechol gene operons (28,36,56,57), where the encoding enzymes catalyze a crucial degradation step channeling the substrate into the The kinetic constants of SalOH, C12O salD , C12O ccaA , MCI salC , and MCI ccaB are indicated. The specific activity (U/g protein) was determined in cell extracts, and the contribution of each of the (chloro)catechol 1,2-dioxygenases or (chloro)muconate cycloisomerases to the total activity against 0.1 mM 4-chlorocatechol or 0.1 mM 3-chloromuconate (A) or against 0.1 mM 4-methylcatechol or 0.1 mM 3-methylmuconate (B) in 5-chlorosalicylate-grown (gray) or 4-methylsalicylategrown (boxed) cells was calculated after enzyme partial purification (given in percent and U/g protein).…”

Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1

“…In contrast to the enzymes from Pseudomonas sp. strain B13, Alcaligenes eutrophus JMP 134, and Pseudomonas aeruginosa RH01, which do not distinguish between 2-chloro-and 3-chloromaleylacetate as substrate (Kaschabek and Reineke 1995;Müller et al 1996), maleylacetate reductase from X. flavus 14p1 converts the 3-chloro-isomer poorly ( Table 2).…”