Genetic Examination of Initial Amino Acid Oxidation and Glutamate Catabolism in the Hyperthermophilic Archaeon Thermococcus kodakarensis
Genetic Examination of Initial Amino Acid Oxidation and Glutamate Catabolism in the Hyperthermophilic Archaeon Thermococcus kodakarensis
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DOI：
10.1128/jb.01979-12
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发表时间：
2013-02
影响因子：
3.2
通讯作者：
Yuusuke Yokooji;Takaaki Sato;S. Fujiwara;T. Imanaka;H. Atomi
中科院分区：
文献类型：
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作者：
Yuusuke Yokooji;Takaaki Sato;S. Fujiwara;T. Imanaka;H. Atomi
摘要热球菌体内氨基酸的分解被认为是通过三个步骤进行的：谷氨酸脱氢酶或氨基转移酶对氨基酸的氧化脱氨，2-氧酸铁氧还蛋白氧化还原酶(KOR)对氨基酸的氧化脱酸，ADP形成的酰辅酶A合成酶(ACS)对酰基辅酶A(CoA)的水解。在这里，我们对柯达卡热球菌中参与谷氨酸分解代谢的酶进行了遗传学检查。Kodakarensis KUW1(ΔpyrFΔtrpE)细胞提取物中氨基酸脱氢酶活性的检测表明，依赖于NADP的GDH活性较高，而依赖于NAD的活性较低。依赖NADP的谷氨酸/丙氨酸/Val/半胱氨酸的活性和依赖NAD的苏氨酸脱氢酶活性也被检测到。KGDH1是一株基因突变株，其苏氨酸脱氢酶活性仅在TK-GDH中检测到，其他活性均依赖于TK-GDH。KGDH1不能在生长依赖于氨基酸分解代谢的介质中生长，这意味着TK-GDH是唯一能释放氨基酸氧化成2-氧酸过程中释放的电子(向NADP+/NAD+)的酶。在含有过量丙酮酸的培养液中，KGDH1表现出正常的生长，但与KUW1相比，其氨基酸分解代谢程度更高，这表明TK-GDH具有抑制氨基酸氧化的作用，并在此条件下发挥合成代谢作用。我们进一步构建了2-羟基戊二酸的中断菌株：铁氧还蛋白氧化还原酶和琥珀酰辅酶A合成酶。与KUW1相比，这两个菌株在两种培养基上都表现出生长缺陷。在这些菌株中没有观察到琥珀酸的产生，这表明这两种酶是柯达卡氏锥虫多种KOR和ACS酶中唯一负责谷氨酸分解代谢的酶。
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.