title: A model for glutathione binding and activation in the fosfomycin resistance protein, FosA.
abstract: The genomically encoded fosfomycin resistance protein from Pseudomonas aeruginosa (FosA(PA)) utilizes Mn(II) and K(+) to catalyze the addition of glutathione (GSH) to C1 of the antibiotic rendering it inactive. Although this protein has been structurally and kinetically characterized with respect to the substrate, fosfomycin, questions remain regarding how the enzyme binds the thiol substrate, GSH. Computational studies have revealed a potential GSH binding site in FosA(PA) that involves six electrostatic or hydrogen-bonding interactions with protein side-chains as well as six additional residues that contribute van der Waals interactions. A strategically placed tyrosine residue, Y39, appears to be involved in the ionization of GSH during catalysis. The Y39F mutant exhibits a 13-fold reduction of catalytic activity (k(cat)=14+/-2s(-1)), suggesting a role in the ionization of GSH. Mutation of five other residues (W34, Q36, S50, K90, and R93) implicated in ionic of hydrogen-bonding interactions resulted in enzymes with reduced catalytic efficiency, affinity for GSH, or both. The mutant enzymes were also found to be less effective resistant proteins in the biological context of Escherichia coli. The more conservative W34H mutant has native-like catalytic efficiency suggesting that the imidazole NH group can replace the indole group of W34 that is important for GSH binding. In the absence of co-crystal structural data with the thiol substrate, these results provide important insights into the role of GSH in catalysis.
authors.affiliation: Department of Chemistry and The Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Rigsby
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Brown
authors.forename: Eric
authors.initials: E
authors.lastname: Dawson
authors.forename: Terry P
authors.initials: TP
authors.lastname: Lybrand
authors.forename: Richard N
authors.initials: RN
authors.lastname: Armstrong
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D004352:Drug Resistance, Microbial; D004789:Enzyme Activation; D005978:Glutathione / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013329:Structure-Activity Relationship
chemical_list: D005978:Glutathione