Generation by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls.

2.50
Hdl Handle:
http://hdl.handle.net/10033/18733
Title:
Generation by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls.
Authors:
Cámara, Beatriz; Seeger, Michael; González, Myriam; Standfuss-Gabisch, Christine; Kahl, Silke; Hofer, Bernd ( 0000-0002-5174-4021 )
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.
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.
Citation:
Generation by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls. 2007, 73 (8):2682-9 Appl. Environ. Microbiol.
Journal:
Applied and environmental microbiology
Issue Date:
Apr-2007
URI:
http://hdl.handle.net/10033/18733
DOI:
10.1128/AEM.02523-06
PubMed ID:
17322323
Type:
Article
Language:
en
ISSN:
0099-2240
Appears in Collections:
publications of the research group microbial interactions and processes (MINP)

Full metadata record

DC FieldValue Language
dc.contributor.authorCámara, Beatrizen
dc.contributor.authorSeeger, Michaelen
dc.contributor.authorGonzález, Myriamen
dc.contributor.authorStandfuss-Gabisch, Christineen
dc.contributor.authorKahl, Silkeen
dc.contributor.authorHofer, Bernden
dc.date.accessioned2008-02-20T14:27:26Zen
dc.date.available2008-02-20T14:27:26Zen
dc.date.issued2007-04en
dc.identifier.citationGeneration by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls. 2007, 73 (8):2682-9 Appl. Environ. Microbiol.en
dc.identifier.issn0099-2240en
dc.identifier.pmid17322323en
dc.identifier.doi10.1128/AEM.02523-06en
dc.identifier.urihttp://hdl.handle.net/10033/18733en
dc.description.abstractRecently, 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.en
dc.language.isoenen
dc.subject.meshBiotransformationen
dc.subject.meshBurkholderiaen
dc.subject.meshDioxygenasesen
dc.subject.meshMetabolic Networks and Pathwaysen
dc.subject.meshOxidation-Reductionen
dc.subject.meshPolychlorinated Biphenylsen
dc.subject.meshProtein Engineeringen
dc.subject.meshPseudomonasen
dc.subject.meshRecombinant Fusion Proteinsen
dc.subject.meshSubstrate Specificityen
dc.titleGeneration by a widely applicable approach of a hybrid dioxygenase showing improved oxidation of polychlorobiphenyls.en
dc.typeArticleen
dc.contributor.departmentLaboratorio 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.en
dc.identifier.journalApplied and environmental microbiologyen
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