eng URI http://hdl.handle.net/10795/2647 ένζυμο βιοτεχνολογία μικροοργανισμός In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity. 10 pp. LOMv1.0 creator LOMv1.0 publisher LOMv1.0 Project Final Beneficiary LOMv1.0 Project Executing Organisation 2012-04-04 Digital Library of the Operational Programme "Education and Lifelong Learning" abstract types Text Glutathione transferases Isoenzymes Enzymes Bacterial GST superfamily Comparative sequence analysis http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034263 919847 application/pdf http://repository.edulll.gr/edulll/bitstream/10795/2647/2/2647_Paper%202.pdf URI http://hdl.handle.net/10795/2647 LOMv1.0creator 2016-04-14T08:42:46Z LOMv1.0validator 2016-04-14T08:42:46Z LOMv1.0 gre no no Copyright EYD-EPEDBM (Operational Programme "Education and Lifelong Learning")