title: A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.
abstract: 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.
authors.affiliation: Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Athens, Greece.
authors.forename: Katholiki
authors.initials: K
authors.lastname: Skopelitou
authors.forename: Prathusha
authors.initials: P
authors.lastname: Dhavala
authors.forename: Anastassios C
authors.initials: AC
authors.lastname: Papageorgiou
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D019295:Computational Biology; D018360:Crystallography, X-Ray; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010802:Phylogeny; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D005982:Glutathione Transferase; D005978:Glutathione