A Glutathione Transferase from <em>Agrobacterium tumefaciens</em> Reveals a Novel Class of Bacterial GST Superfamily

<div><p>In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium <em>Agrobacterium tumefaciens</em> C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from <em>A. tumefaciens</em> C58 (<em>Atu</em>3701) 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 <em>Atu</em>GSTH1-1) was cloned and expressed in <em>E. coli</em> and its structural and catalytic properties were investigated. Functional analysis showed that <em>Atu</em>GSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of <em>Atu</em>GSTH1-1 was determined at 1.4 Å resolution in complex with <em>S</em>-(p-nitrobenzyl)-glutathione (Nb-GSH). Although <em>Atu</em>GSTH1-1 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 <em>Atu</em>GSTH1-1 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.</p>