title: Recombinant expression of a unique chloromuconolactone dehalogenase ClcF from Rhodococcus opacus 1CP and identification of catalytically relevant residues by mutational analysis.
abstract: Chloromuconolactone dehalogenase ClcF plays a unique role in 3-chlorocatechol degradation by Rhodococcus opacus 1CP by compensating the inability of its chloromuconate cycloisomerase ClcB2 to dechlorinate the chemically stable cycloisomerization product (4R,5S)-5-chloromuconolactone (5CML). High sequence similarities showed relatedness of ClcF to muconolactone isomerases (MLIs, EC 5.3.3.4) of the 3-oxoadipate pathway. Although both enzyme types share the ability to dechlorinate 5CML, comparison of kcat/Km indicated a significant extent of specialization of ClcF for dechlorination. This assumption was substantiated by an almost complete inability of ClcF to convert (4S)-muconolactone and the exclusive formation of cis-dienelactone from 5CML. Mutational analysis of ClcF by means of variants E27D, E27Q, Y50A, N52A, and A89S indicated relevance of some highly conserved residues for substrate binding and catalysis. Based on the putative isomerization mechanism of MLI, evidence was provided for a role of E27 in initial proton abstraction as well as of Y50 and N52 in substrate binding. In case of N52 substrate binding is likely to occur to the carboxylic group of 5CML as indicated by a significant change of product specificity. Expression in Escherichia coli BL21-CP(DE)-RIL followed by a three-step purification procedure with heat treatment is a convenient strategy to obtain recombinant ClcF and variants thereof.
authors: Environmental Microbiology, TU Bergakademie Freiberg, Freiberg, Germany.
authors: Janosch A D
authors: JA
authors: Gröning
authors: Christian
authors: C
authors: Roth
authors: Stefan R
authors: SR
authors: Kaschabek
authors: Norbert
authors: N
authors: Sträter
authors: Michael
authors: M
authors: Schlömann
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D055162:Biocatalysis; D019748:Carbon-Carbon Double Bond Isomerases / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D020118:Cupriavidus necator / Q000201:enzymology; D004252:DNA Mutational Analysis; D015870:Gene Expression; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D007783:Lactones; D011994:Recombinant Proteins; D006867:Hydrolases; D019748:Carbon-Carbon Double Bond Isomerases; C100087:muconolactone delta-isomerase