Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase.
GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.
Federici
Luca
L
Ce.S.I. (Centro Studi sull'Invecchiamento), Fondazione Università di Chieti G. d'Annunzio, Via dei Vestini 31, 66013 Chieti, Italy.
Masulli
Michele
M
Bonivento
Daniele
D
Di Matteo
Adele
A
Gianni
Stefano
S
Favaloro
Bartolo
B
Di Ilio
Carmine
C
Allocati
Nerino
N
D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D020621:Ochrobactrum anthropi / Q000201:enzymology* / Q000235:genetics; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature
D012694:Serine; D005982:Glutathione Transferase