Chlorocatechol 1,2‐Dioxygenase from Rhodococcus Erythropolis 1CP

Abstract

Chlorocatechol 1,2-dioxygenase from Rhodococcus erythropolis 1 CP was purified to homogeneity. In contrast to chlorocatechol 1,2-dioxygenase from Gram-negative strains which have a very broad substrate tolerance, the Rhodococcus enzyme was relatively more specific and had a distinct preference for 4-substituted catechols. Protein and metal analysis indicate an unusual stoichiometry of one atom each of iron and manganese/64-kDa homodimer. The N-terminal amino acid sequence (27 residues) of the Rhodococcus chlorocatechol 1,2-dioxygenase was determined and exhibited 15 -22% identity to the published sequences of catechol 1,2-dioxygenases and other chlorocatechol 1,2-dioxygenases. Antiserum was raised in rabbits and antibodies against Rhodococcus chlorocatechol 1,2-dioxygenase were affinity purified. Dot-blot analysis revealed a very weak reaction between the antibodies and partially purified chlorocatechol 1,2-dioxygenases from Alcaligenes eutrophus JMPl34 and Pseudomonas putida 87. No reaction between these antibodies and above enzymes was observed using Western blotting. Kinetic and immunochemical data as well as comparison of subunit molecular mass and suggest that the Rhodococcus enzyme differs significantly from the known highly similar chlorocatechol 1,2-dioxygenases of Gram-negative strains and seems to be only distantly related to them.The ability to catalyze transformation of different monochloroaromatic compounds (chlorophenols, chloroanilines and chlorobenzoates) to the corresponding chlorinated catechols has been shown to be quite common among the representatives of the nocardiafoxm genus Rhodococcus (Golovlev and Eroshina, 1982;

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

“…In particular, the 1,2-CCDs from Pseudomonas putida pAC27, Ralstonia eutropha pJP4, Pseudomonas chlororaphis RW71 and the 3-chlorocatechol specific 1,2-CCD from R. opacus CP1 (same strain expressing the present enzyme) exhibit higher activities toward 3-chlorocatechol than Rho 1,2-CCD. Possible reasons for the different observed specificities could be the replacement of Phe-78, a residue expected to interact with substituents in position 3, with Tyr, and in the most internal side of the cavity Ala-53, which is exchanged with Val in all the other 1,2-CCDs described (35,36,41,71). Furthermore, Cys-223 and 224, suggested to be important for interacting with chlorine substituents, are generally conserved in CCDs, as shown in Fig.…”

“…The residues surrounding the ring of the benzoate molecule and supposed to be involved in the correct positioning of the aromatic substrate are Leu-49, Asp-52, Ala-53, Ile-74, Gly-76, Pro-77, Phe-78, Gln-210, and Cys-224. The present enzyme is able to catalyze the ring opening of a variety of substituted catechols (41). We attempted to dock 3-or 4-chlorocatechols into the active site.…”

“…Protein Preparation, Crystallization, and Data Collection-Rho 1,2-CCD was purified from R. opacus 1CP as reported previously (41). The enzyme was crystallized at 293 K using the sitting drop vapor diffusion method from a solution containing 1.6 M ammonium sulfate, 0.1 M sodium chloride, 100 mM Tris-HCl, pH 7.5, and 5-15% glycerol (53).…”

“…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).…”

Metabolism of 5‐Chlorosubstituted Muconolactones

“…The ortho-cleavage enzymes involved in the metabolism of catechols differ significantly in substrate specificity ; classification into chlorocatechol 1,2-dioxygenases of broad substrate specificity and catechol 1,2-dioxygenases of restricted specificity, does not cover the whole set of enzymes as distantly related enzymes of intermediate specificity have recently been described (Maltseva et al, 1994a). A much more extreme diversity has been shown for the cycloisomenzing enzymes acting on muconates.…”