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Directed evolution of a non-heme-iron-dependent extradiol catechol dioxygenase: Identification of mutants with intradiol oxidative cleavage activity
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Schlosrich, Janne, Eley, Kirstin L., Crowley, Patrick J. and Bugg, Tim (2006) Directed evolution of a non-heme-iron-dependent extradiol catechol dioxygenase: Identification of mutants with intradiol oxidative cleavage activity. CHEMBIOCHEM, 7 (12). pp. 1899-1908. doi:10.1002/cbic.200600296 ISSN 1439-4227.
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Official URL: http://dx.doi.org/10.1002/cbic.200600296
Abstract
The non-heme-iron (II)-dependent extradiol catechol dioxygenases catalyse the oxidative cleavage of substituted catechols found on bacterial aromatic degradation pathways. The reaction mechanism of the extradiol dioxygenases is believed to proceed through the same proximal hydroperoxide intermediate as the iron(III)-dependant intradiol catechol dioxygenases. Directed evolution was carried out on members of the class III extradiol catechol dioxygenases, by using 1) error-prone polymerase chain reaction, 2) a primar-based cross-over method; the mutant dioxygenases were then screened for their ability to process a range of substituted catechols. Several mutant enzymes were found to show higher activity towards certain substituted catechols, including 4-chloro-catechol, and higher affinity for the iron(II) cofactor. Two mutants isolated from error-prone PCR of Escherichia coli MhpB (mutants R215W and K273R) were found to produce a mixture of extradiol and intradiol cleavage products, as detected by GC-MS and H-1 NMR spectroscopy. The residue corresponding to K273 in pro-tocatechuate 4,5-dioxygenase (LigAB), Val244, is located approximately 12 angstrom from the iron (II) centre, but close to the putative dioxygen channel; R215 id found on a sequence loop not present in LigB.
Item Type: | Journal Article | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Journal or Publication Title: | CHEMBIOCHEM | ||||
Publisher: | WILEY-V C H VERLAG GMBH | ||||
ISSN: | 1439-4227 | ||||
Official Date: | December 2006 | ||||
Dates: |
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Volume: | 7 | ||||
Number: | 12 | ||||
Number of Pages: | 10 | ||||
Page Range: | pp. 1899-1908 | ||||
DOI: | 10.1002/cbic.200600296 | ||||
Publication Status: | Published |
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