title: Characterization of the hydrophobic substrate-binding site of the bacterial beta class glutathione transferase from Proteus mirabilis.
abstract: Since their discovery, bacterial glutathione (GSH)transferases have been characterized in terms of their ability to catalyse a variety of different reactions on a large set of toxic molecules of xenobiotic or endobiotic origin. Furthermore the contribution of different residues in the GSH-binding site to GSH activation has been extensively investigated. Little is known, however, about the contribution to catalysis and overall stability of single residues shaping the hydrophobic co-substrate binding site (H-site). Here we tackle this problem by site-directed mutagenesis of residues facing the H-site in the bacterial beta class GSH transferase from Proteus mirabilis. We investigate the behaviour of these mutants under a variety of conditions and analyse their activity against several co-substrates, representative of the different reactions catalyzed by bacterial GSH transferases. Our work shows that mutations at the H-site can be used to modulate activity at the level of the different catalytic mechanisms operating on the chosen substrates, each mutation showing a different fingerprint. This work paves the way for future studies aimed at improving the catalytic properties of beta class GSH transferases against selected substrates for bioremediation purposes.
authors.affiliation: Dipartimento di Scienze Biomediche, Università G. d'Annunzio, Via dei Vestini 31, I-66013 Chieti, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Federici
authors.forename: Michele
authors.initials: M
authors.lastname: Masulli
authors.forename: Carmine
authors.initials: C
authors.lastname: Di Ilio
authors.forename: Nerino
authors.initials: N
authors.lastname: Allocati
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001555:Benzene Derivatives / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D004795:Enzyme Stability; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D011513:Proteus mirabilis / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D001555:Benzene Derivatives; D005982:Glutathione Transferase; D005978:Glutathione