title: Purification and characterization of Clostridium difficile glutamate dehydrogenase.
abstract: Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.
authors.affiliation: Department of Biochemistry & Nutrition, Virginia Polytechnic Institute & State University, Blacksburg 24061-0308.
authors.forename: B M
authors.initials: BM
authors.lastname: Anderson
authors.forename: C D
authors.initials: CD
authors.lastname: Anderson
authors.forename: R L
authors.initials: RL
authors.lastname: Van Tassell
authors.forename: D M
authors.initials: DM
authors.lastname: Lyerly
authors.forename: T D
authors.initials: TD
authors.lastname: Wilkins
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D016360:Clostridioides difficile / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D046911:Macromolecular Substances; D009243:NAD; D005969:Glutamate Dehydrogenase