TY - JOUR
T1 - Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis
AU - Wakamatsu, Taisuke
AU - Higashi, Chisato
AU - Ohmori, Taketo
AU - Doi, Katsumi
AU - Ohshima, Toshihisa
N1 - Funding Information:
Acknowledgments This work was supported in part by Grant-in-Aid for Scientific Research 22248010 (to T.O.) from the Ministry of Education, Science, Sports and Culture of Japan.
PY - 2013/5
Y1 - 2013/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84876686889&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84876686889&partnerID=8YFLogxK
U2 - 10.1007/s00792-013-0527-7
DO - 10.1007/s00792-013-0527-7
M3 - Article
C2 - 23508687
AN - SCOPUS:84876686889
SN - 1431-0651
VL - 17
SP - 379
EP - 389
JO - Extremophiles
JF - Extremophiles
IS - 3
ER -