title: Intersubunit disulfide interactions play a critical role in maintaining the thermostability of glucose-6-phosphate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus.
abstract: Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t1/2 = 24 hr at 90 degrees C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine 184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines. The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90 degrees C. The same decrease in half-life was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability of the enzyme.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
authors.forename: Manjula
authors.initials: M
authors.lastname: Nakka
authors.forename: Ramesh B
authors.initials: RB
authors.lastname: Iyer
authors.forename: Leonidas G
authors.initials: LG
authors.lastname: Bachas
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides; D004795:Enzyme Stability; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins; D040681:Structural Homology, Protein
chemical_list: D004220:Disulfides; D021122:Protein Subunits; D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase; D003545:Cysteine