Structural basis for the substrate specificity and the absence of dehalogenation activity in 2-chloromuconate cycloisomerase from Rhodococcus opacus 1CP

• Published in: Biochimica et biophysica acta 2014-09, Vol.1844 (9), p.1541-1549
• Main Authors: Kolomytseva, Marina, Ferraroni, Marta, Chernykh, Alexey, Golovleva, Ludmila, Scozzafava, Andrea
• Format: Article
• Language: English
• Subjects: 4-Butyrolactone - analogs & derivatives; 4-Butyrolactone - chemistry; 4-Butyrolactone - metabolism; Adipates - chemistry; Adipates - metabolism; Aromatic catabolism; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Catalytic Domain; Catechols - chemistry; Catechols - metabolism; Chlorophenols - chemistry; Chlorophenols - metabolism; Crystallography, X-Ray; Dehalogenation; Histidine - chemistry; Histidine - metabolism; Intramolecular Lyases - chemistry; Intramolecular Lyases - isolation & purification; Intramolecular Lyases - metabolism; Lactones - chemistry; Lactones - metabolism; Modified ortho-pathway; Molecular Docking Simulation; Muconate; Protein Multimerization; Pseudomonas putida - chemistry; Pseudomonas putida - enzymology; Rhodococcus - chemistry; Rhodococcus - enzymology; Sorbic Acid - analogs & derivatives; Sorbic Acid - chemistry; Sorbic Acid - metabolism; Structural Homology, Protein; Structure-Activity Relationship; Substrate Specificity; X-ray crystallography
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2014.04.008

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. •The X-ray structure of 2-chloromuconate cyclomerase (2-CMCI) was solved.•Molecular docking calculation on 2-CMCI was performed.•The molecular determinants for its narrow substrate specificity are discussed.•A residue responsible of its lack of dehalogenation activity was identified.