Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate
Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate
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DOI：
10.1111/j.1365-2958.2011.07762.x
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发表时间：
2011-09-01
影响因子：
3.6
通讯作者：
Imanaka, Tadayuki
中科院分区：
文献类型：
--
作者：
Matsubara, Kohei;Yokooji, Yuusuke;Imanaka, Tadayuki
在经典的Embden-Meyerhof （EM）糖酵解途径中，甘油醛3-磷酸（GAP）与3-磷酸甘油酸（3-PGA）之间的转化是通过磷酸化GAP脱氢酶（GAPDH）和磷酸甘油酸激酶（PGK）可逆催化的。在Euryarchaeota kodakarensis热球菌和Pyrococcus furiosus中，存在一个额外的编码GAP的基因：铁氧还蛋白氧化还原酶（GAPOR）和一个类似于非磷酸化GAP脱氢酶（GAPN）的基因。为了确定连接GAP和3-PGA的三条途径的生理作用，我们分别破坏了T. kodakarensis的GAPOR、GAPN、GAPDH和PGK基因（分别为gor、GAPN、GAPDH和PGK）。Delta gor菌株在糖酵解条件下没有生长，证实了其在糖酵解过程中产生还原铁氧还蛋白以产生能量的功能。令人惊讶的是，δ gapN细胞在糖酵解条件下也不能生长，这表明gapN在这些条件下提供NADPH中起关键作用。gor和gapN的破坏对糖异生生长没有影响。Delta gapDH和Delta pgk菌株的生长实验表明，与经典EM途径中的对应菌株不同，gapDH / pgk仅在糖异生中起主要作用。生化分析表明，T. kodakarensis GAPN不识别除D-GAP以外的醛类底物，首选NADP（+）作为辅助因子，并被葡萄糖1-磷酸显著激活。
In the classical Embden-Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP: ferredoxin oxidoreductase (GAPOR) and a gene similar to non-phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3-PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T. kodakarensis. The Delta gor strain displayed no growth under glycolytic conditions, confirming its proposed function to generate reduced ferredoxin for energy generation in glycolysis. Surprisingly, Delta gapN cells also did not grow under glycolytic conditions, suggesting that GAPN plays a key role in providing NADPH under these conditions. Disruption of gor and gapN had no effect on gluconeogenic growth. Growth experiments with the Delta gapDH and Delta pgk strains indicated that, unlike their counterparts in the classical EM pathway, GAPDH/PGK play a major role only in gluconeogenesis. Biochemical analyses indicated that T. kodakarensis GAPN did not recognize aldehyde substrates other than D-GAP, preferred NADP(+) as cofactor and was dramatically activated with glucose 1-phosphate.