NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae -: Purification, kinetic properties, and physiological roles
NADP-glutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae -: Purification, kinetic properties, and physiological roles
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DOI：
10.1074/jbc.m107986200
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发表时间：
2001-11-23
影响因子：
4.8
通讯作者：
González, A
中科院分区：
文献类型：
--
作者：
DeLuna, A;Avendaño, A;González, A
在酿酒酵母中，由GDH1和GDH3编码的两个NADP（+）依赖性谷氨酸脱氢酶（NADP- gdhs）催化铵和a-酮戊二酸合成谷氨酸。gdh2编码的NAD（+）依赖性谷氨酸脱氢酶降解谷氨酸，产生铵和a-酮戊二酸。直到最近，人们才认为酿酒葡萄球菌中只存在一种生物合成的NADP-GDH。这一事实阻碍了理解每个同工酶的生理作用和参与谷氨酸生物合成的a-酮戊二酸通道的机制。在本研究中，我们纯化并表征了GDH1-和gdh3编码的NADP-GDHs；它们表现出不同的变构特性和a-酮戊二酸利用率。对这些蛋白相对水平的分析表明，GDH1和GDH3的表达受到不同的调控，并取决于碳源的性质。此外，对缺乏或过表达GDH1或GDH3的突变体的生理研究表明，这些基因发挥着非冗余的生理作用。我们的研究结果表明，在发酵和呼吸条件下，GDH1-、GDH3-和gdh2编码酶的协调调节导致谷氨酸的生物合成和a-酮戊二酸的平衡利用。讨论了复制NADP-GDH通路在兼性代谢适应中的可能相关性。
In the yeast Saccharomyces cerevisiae, two NADP(+)-dependent glutamate dehydrogenases (NADP-GDHs) encoded by GDH1 and GDH3 catalyze the synthesis of glutamate from ammonium and a-ketoglutarate. The GDH2-encoded NAD(+)-dependent glutamate dehydrogenase degrades glutamate producing ammonium and a-ketoglutarate. Until very recently, it was considered that only one biosynthetic NADP-GDH was present in S. cerevisiae. This fact hindered understanding the physiological role of each isoenzyme and the mechanisms involved in a-ketoglutarate channeling for glutamate biosynthesis. In this study, we purified and characterized the GDH1- and GDH3-encoded NADP-GDHs; they showed different allosteric properties and rates of a-ketoglutarate utilization. Analysis of the relative levels of these proteins revealed that the expression of GDH1 and GDH3 is differentially regulated and depends on the nature of the carbon source. Moreover, the physiological study of mutants lacking or overexpressing GDH1 or GDH3 suggested that these genes play nonredundant physiological roles. Our results indicate that the coordinated regulation of GDH1-, GDH3-, and GDH2-encoded enzymes results in glutamate biosynthesis and balanced utilization of a-ketoglutarate under fermentative and respiratory conditions. The possible relevance of the duplicated NADP-GDH pathway in the adaptation to facultative metabolism is discussed.