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PMID:22496785
| Citation |
Skopelitou, K, Dhavala, P, Papageorgiou, AC and Labrou, NE (2012) A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily. PLoS ONE 7:e34263 |
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| 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. |
| Links |
PubMed PMC3319563 Online version:10.1371/journal.pone.0034263 |
| Keywords |
Agrobacterium tumefaciens/enzymology; Amino Acid Sequence; Binding Sites; Catalysis; Computational Biology; Crystallography, X-Ray; Glutathione/metabolism; Glutathione Transferase/chemistry; Glutathione Transferase/genetics; Glutathione Transferase/metabolism; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation/genetics; Phylogeny; Protein Conformation; Sequence Homology, Amino Acid |
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0004096: catalase activity |
ECO:0000315: |
F |
Table 4 shows AtuGSTH1-1 structure is similar to GST proteins whose active site interacts sulfhydrly groups in GSH to produce thiolate anions. However the gene was missing the functional side chain for this activity.They created alanine mutants to see if they catalyzed reactions on the substrates CDNB and cumene hydroperoxide. Table three results showed that residues at Phen22 and Ser25 where crucial in the cumene hydroperoxide pathway. Indicating that AtuGSTH1-1 natural substrate is the breakdown of hydroperoxides. Table 1 provides additional evidence showing that AtuGSTH1-1 has a high catalytic efficiency on electrophile substrates of cumene peroxide and tert-butyl peroxide. |
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