Guehler Biochemistry Research Laboratory, Department of Chemistry, Augustana College, Rock Island, Illinois.
Biologie intégrative du Métabolisme Lipidique, Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.
Yeast. 2020 Jan;37(1):103-115. doi: 10.1002/yea.3425. Epub 2019 Aug 8.
Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet little is known about these enzymes in the oleaginous yeast Yarrowia lipolytica. The YALI0F17820g and YALI0E09603g genes, encoding potential GDH enzymes in this organism, were examined. Heterologous expression in gdh-null Saccharomyces cerevisiae and examination of Y. lipolytica strains carrying gene deletions demonstrate that YALI0F17820g (ylGDH1) encodes a NADP-dependent GDH whereas YALI0E09603g (ylGDH2) encodes a NAD-dependent GDH enzyme. The activity encoded by these two genes accounts for all measurable GDH activity in Y. lipolytica. Levels of the two enzyme activities are comparable during logarithmic growth on rich medium, but the NADP-ylGDH1p enzyme activity is most highly expressed in stationary and nitrogen starved cells by threefold to 12-fold. Replacement of ammonia with glutamate causes a decrease in NADP-ylGdh1p activity, whereas NAD-ylGdh2p activity is increased. When glutamate is both carbon and nitrogen sources, the activity of NAD-ylGDH2p becomes dominant up to 18-fold compared with that of NADP-ylGDH1p. Gene deletion followed by growth on different carbon and nitrogen sources shows that NADP-ylGdh1p is required for efficient nitrogen assimilation whereas NAD-ylGdh2p plays a role in nitrogen and carbon utilization from glutamate. Overexpression experiments demonstrate that ylGDH1 and ylGDH2 are not interchangeable. These studies provide a vital basis for future consideration of how these enzymes function to facilitate energy and nitrogen homeostasis in Y. lipolytica.
谷氨酸脱氢酶（GDH）对细胞的氮和能量平衡至关重要。然而，关于产油酵母解脂耶氏酵母中的这些酶知之甚少。本文研究了该生物体中编码潜在 GDH 酶的 YALI0F17820g 和 YALI0E09603g 基因。在 gdh 缺失的酿酒酵母中的异源表达和携带基因缺失的 Y. lipolytica 菌株的研究表明，YALI0F17820g（ylGDH1）编码 NADP 依赖性 GDH，而 YALI0E09603g（ylGDH2）编码 NAD 依赖性 GDH 酶。这两个基因编码的酶活性占解脂耶氏酵母中所有可测量的 GDH 活性。在富培养基上对数生长期，两种酶活性水平相当，但在静止和氮饥饿细胞中，NADP-ylGDH1p 酶活性的表达水平是其三倍到十二倍。用谷氨酸替代氨会导致 NADP-ylGdh1p 活性降低，而 NAD-ylGdh2p 活性增加。当谷氨酸既是碳源又是氮源时，NAD-ylGDH2p 的活性与 NADP-ylGDH1p 的活性相比增加了 18 倍。不同碳源和氮源生长后的基因缺失实验表明，NADP-ylGdh1p 是有效氮同化所必需的，而 NAD-ylGdh2p 在从谷氨酸中利用氮和碳方面发挥作用。过表达实验表明，ylGDH1 和 ylGDH2 不能互换。这些研究为进一步研究这些酶在解脂耶氏酵母中如何发挥作用以促进能量和氮平衡提供了重要基础。
