dienelactone and 5-chloromuconolactone

2-Chloromuconate cycloisomerase from the Gram-positive bacterium Rhodococcus opacus 1CP (Rho-2-CMCI) is an enzyme of a modified ortho-pathway, in which 2-chlorophenol is degraded using 3-chlorocatechol as the central intermediate. In general, the chloromuconate cycloisomerases catalyze not only the cycloisomerization, but also the process of dehalogenation of the chloromuconate to dienelactone. However Rho-2-CMCI, unlike the homologous enzymes from the Gram-negative bacteria, is very specific for only one position of the chloride on the substrate chloromuconate. Furthermore, Rho-2-CMCI is not able to dehalogenate the 5-chloromuconolactone and therefore it cannot generate the dienelactone. The crystallographic structure of the homooctameric Rho-2-CMCI was solved by molecular replacement using the coordinates of the structure of chloromuconate cycloisomerase from Pseudomonas putida PRS2000. The structure was analyzed and compared to the other already known structures of (chloro)muconate cycloisomerases. In addition to this, molecular docking calculations were carried out, which allowed us to determine the residues responsible for the high substrate specificity and the lack of dehalogenation activity of Rho-2-CMCI. Our studies highlight that a histidine, located in a loop that closes the active site cavity upon the binding of the substrate, could be related to the dehalogenation inability of Rho-2-CMCI and in general of the muconate cycloisomerases.

Topics: 4-Butyrolactone; Adipates; Bacterial Proteins; Catalytic Domain; Catechols; Chlorophenols; Crystallography, X-Ray; Histidine; Intramolecular Lyases; Lactones; Molecular Docking Simulation; Protein Multimerization; Pseudomonas putida; Rhodococcus; Sorbic Acid; Structural Homology, Protein; Structure-Activity Relationship; Substrate Specificity

The actinobacterium Rhodococcus opacus 1CP possesses a so far unique variant of the modified 3-oxoadipate pathway for 3-chlorocatechol degradation. One important feature is the novel dehalogenase ClcF, which converts (4R,5S)-5-chloromuconolactone to E-dienelactone. ClcF is related to muconolactone isomerase (MLI, EC 5.3.3.4). The enzyme has a ferredoxin-type fold and forms a homodecamer of 52-symmetry, typical for the MLI family. The active site is formed by residues from two monomers. The complex structure of an E27A variant with bound substrate in conjunction with mutational studies indicate that E27 acts as the proton acceptor in a univalent single-base syn-dehydrohalogenation mechanism. Despite the evolutionary specialization of ClcF, the conserved active-site structures suggest that the proposed mechanism is representative for the MLI family. Furthermore, ClcF represents a novel type of dehalogenase based on an isomerase scaffold.

Topics: 4-Butyrolactone; Amino Acid Sequence; Bacterial Proteins; Catalysis; Crystallization; Crystallography, X-Ray; DNA Mutational Analysis; Hydrolases; Lactones; Molecular Sequence Data; Protein Structure, Tertiary; Rhodococcus; Sequence Alignment

5-Chloromuconolactone dehalogenase (5-CMLD) is a unique enzyme that catalyzes the conversion of 5-chloromuconolactone into cis-dienelactone in the new modified ortho-pathway of the 3-chlorocatechol degradation by Rhodococcus opacus 1CP. In all other known chlorocatechol pathways the dehalogenation is a spontaneous secondary reaction of the unstable chloromuconate intermediate following the lactonization process catalyzed by the muconate cycloisomerases. The crystallographic structure of the decameric 5-CMLD was solved by Molecular Replacement, using the coordinates of the low resolution structure of the highly homologous muconolactone isomerase, an enzyme of the conventional ortho-pathway. Muconolactone isomerase catalyzes the endocyclic rearrangement of the double bond within the lactone ring of muconolactone to yield 3-oxoadipate enol lactone. Although both 5-CMLD and muconolactone isomerase share the ability to dechlorinate 5-chloromuconolactone, 5-CMLD shows a significant degree of specialization, having lost the capacity to convert its original substrate muconolactone. The active site of 5-CMLD was previously hypothesized to reside in a deep pocket at the interface of two different subunits, on the basis of a muconolactone isomerase structure analysis. In this study we also performed molecular docking calculations that confirmed these previous findings, and allowed us furthermore to determine the residues involved in the catalytic process.

Topics: 4-Butyrolactone; Amino Acid Sequence; Bacterial Proteins; Biocatalysis; Carbon-Carbon Double Bond Isomerases; Catalytic Domain; Crystallography, X-Ray; Hydrolases; Lactones; Molecular Docking Simulation; Molecular Sequence Data; Protein Structure, Tertiary; Rhodococcus; Sequence Alignment