title: Genetic examination of initial amino acid oxidation and glutamate catabolism in the hyperthermophilic archaeon Thermococcus kodakarensis.
abstract: Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T. kodakarensis KUW1 (ΔpyrF ΔtrpE) revealed high NADP-dependent GDH activity, along with lower levels of NAD-dependent activity. NADP-dependent activities toward Gln/Ala/Val/Cys and an NAD-dependent threonine dehydrogenase activity were also detected. In KGDH1, a gene disruption strain of T. kodakarensis GDH (Tk-GDH), only threonine dehydrogenase activity was detected, indicating that all other activities were dependent on Tk-GDH. KGDH1 could not grow in a medium in which growth was dependent on amino acid catabolism, implying that Tk-GDH is the only enzyme that can discharge the electrons (to NADP(+)/NAD(+)) released from amino acids in their oxidation to 2-oxoacids. In a medium containing excess pyruvate, KGDH1 displayed normal growth, but higher degrees of amino acid catabolism were observed compared to those for KUW1, suggesting that Tk-GDH functions to suppress amino acid oxidation and plays an anabolic role under this condition. We further constructed disruption strains of 2-oxoglutarate:ferredoxin oxidoreductase and succinyl-CoA synthetase. The two strains displayed growth defects in both media compared to KUW1. Succinate generation was not observed in these strains, indicating that the two enzymes are solely responsible for Glu catabolism among the multiple KOR and ACS enzymes in T. kodakarensis.
authors: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.
authors: Yuusuke
authors: Y
authors: Yokooji
authors: Takaaki
authors: T
authors: Sato
authors: Shinsuke
authors: S
authors: Fujiwara
authors: Tadayuki
authors: T
authors: Imanaka
authors: Haruyuki
authors: H
authors: Atomi
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D019712:Thermococcus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D019843:Archaeal Proteins; D018698:Glutamic Acid; D007658:Ketone Oxidoreductases; C023096:2-oxoglutarate synthase; D005969:Glutamate Dehydrogenase