Boles E, Lehnert W, Zimmermann F K
Institut für Mikrobiologie, Technische Hochschule Darmstadt, Germany.
Eur J Biochem. 1993 Oct 1;217(1):469-77. doi: 10.1111/j.1432-1033.1993.tb18266.x.
Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose. These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase. GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase. Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase. Moreover, this suppression required functional mitochondria. It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway. Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH. Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria. Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH.
酿酒酵母的磷酸葡萄糖异构酶pgi1缺失突变体不能以葡萄糖作为唯一碳源生长，甚至会受到葡萄糖的抑制。这些生长缺陷可以通过在多拷贝质粒上过量表达编码NAD依赖型谷氨酸脱氢酶的结构基因GDH2来抑制。GDH2编码一种含有1092个氨基酸的蛋白质，该蛋白质位于第十二号染色体上，与粗糙脉孢菌的NAD谷氨酸脱氢酶具有高度的序列相似性。在缺失葡萄糖-6-磷酸脱氢酶基因ZWF1或编码NADPH依赖型谷氨酸脱氢酶的基因GDH1的菌株中，过量表达GDH2对pgi1缺失的抑制作用消失。此外，这种抑制需要功能性线粒体。有人提出，pgi1缺失突变体在葡萄糖上的生长缺陷是由于戊糖磷酸途径氧化反应中作为辅酶所需的NADP迅速耗尽。NAD依赖型谷氨酸脱氢酶的过量表达导致谷氨酸与NADH生成2-氧代戊二酸的高效转化，而2-氧代戊二酸可以通过NADPH依赖型谷氨酸脱氢酶消耗NADPH再转化回谷氨酸。因此，NAD依赖型谷氨酸脱氢酶的过量表达导致2-氧代戊二酸和谷氨酸之间的底物循环，通过NAD到NADH的偶联转化从NADPH中恢复NADP，而NADH可以在线粒体中被氧化。此外，添加已知可降低NADPH水平的氧化剂可以满足抑制磷酸葡萄糖异构酶缺陷对增加NADPH消耗的需求。
