title: Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis.
abstract: Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail. Although both purified GDHs had a hexameric structure and neither exhibited allosteric regulation, they showed different coenzyme specificities: one was specific for NAD(+), the other for NADP(+) and different heat activation mechanisms. In addition, there was little difference in the kinetic constants, optimal temperature, thermal stability, optimal pH and pH stability between the two enzymes. The overall sequence identity between the two proteins was very high (81%), but was not high in the region recognizing the 2' position of the adenine ribose moiety, which is responsible for coenzyme specificity. This is the first report on the identification of two GDHs with different coenzyme specificities from a single hyperthermophilic archaeon and the definition of their basic in vitro properties.
authors.affiliation: Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. taisuke@agr.kyushu-u.ac.jp
authors.forename: Taisuke
authors.initials: T
authors.lastname: Wakamatsu
authors.forename: Chisato
authors.initials: C
authors.lastname: Higashi
authors.forename: Taketo
authors.initials: T
authors.lastname: Ohmori
authors.forename: Katsumi
authors.initials: K
authors.lastname: Doi
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010802:Phylogeny; D055503:Protein Multimerization; D055550:Protein Stability; D041543:Pyrobaculum / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase