title: Generation by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls.
abstract: Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology 149:1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the bphA gene cluster of Burkholderia xenovorans LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from Pseudomonas sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at meta and para carbons. It attacked ortho-, meta-, and para-chlorinated rings with comparable efficiencies. It gave significantly higher yields in ortho,meta-dioxygenation of recalcitrant congeners containing a doubly ortho-chlorinated ring. While the parental enzyme yielded mainly unproductive meta, para dioxygenation of 2,5,4'-CB, the hybrid predominantly converted this congener into an ortho,meta-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.
authors.affiliation: Laboratorio de Microbiología Molecular y Biotechnología Ambiental, Departamento de Química and Millennium Nucleus of Microbial Ecology and Environmental Microbiology and Biotechnology, Universidad Téchnica Federico Santa María, Valparaíso, Chile.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Cámara
authors.forename: Michael
authors.initials: M
authors.lastname: Seeger
authors.forename: Myriam
authors.initials: M
authors.lastname: González
authors.forename: Christine
authors.initials: C
authors.lastname: Standfuss-Gabisch
authors.forename: Silke
authors.initials: S
authors.lastname: Kahl
authors.forename: Bernd
authors.initials: B
authors.lastname: Hofer
mesh_terms: D001711:Biotransformation; D019117:Burkholderia / Q000201:enzymology / Q000235:genetics*; D049308:Dioxygenases / Q000235:genetics* / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000235:genetics / Q000502:physiology; D010084:Oxidation-Reduction; D011078:Polychlorinated Biphenyls / Q000378:metabolism*; D015202:Protein Engineering; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D011078:Polychlorinated Biphenyls; D049308:Dioxygenases