Lysine245 plays a crucial role in stability and function of glycerol 3-phosphate dehydrogenase (Gpd1) in Saccharomyces cerevisiae,Journal of Cellular Biochemistry

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Lysine245 plays a crucial role in stability and function of glycerol 3-phosphate dehydrogenase (Gpd1) in Saccharomyces cerevisiae
Journal of Cellular Biochemistry ( IF 3.0 ) Pub Date : 2021-08-08 , DOI: 10.1002/jcb.30125
Anusha R Pallapati ₁ , Sri D Sirigiri ₁ , Swati Jain ₁ , Vamsi Ratnala ₁ , Ipsita Roy ₁

Affiliation

Glycerol 3-phosphate dehydrogenase (Gpd1 isoform) catalyzes the rate limiting step of glycerol synthesis and is a critical component of the osmo-responsive machinery in yeast. The three-dimensional structure of the enzyme is similar to the enzyme from many other organisms, including humans. A recent study with the human enzyme has proposed K120 (K152 in yeast) to be in the correct orientation for catalysis; K204 (K245 in yeast) is out of plane and is not a participant in the catalytic cycle. The current work was carried out to establish the role of K245 in the catalytic cycle of yeast Gpd1. K245A mutant was found to possess lower catalytic activity. Osmotically stressed cells expressing Gpd1 (K245A) showed no change in intracellular glycerol as compared with wild-type cells which showed ~60% increase. Fluorescence microscopy, native polyacrylamide gel electrophoresis (PAGE) analysis, fluorescence spectroscopy, and Thioflavin T spectrofluorimetry showed a relatively unstable, aggregation- and degradation-prone conformation for the mutant. In silico studies showed an aggregation “hotspot” around K245. This study establishes the requirement of K245 for conformational stability and functional adaptation of Gpd1 in Saccharomyces cerevisiae.

中文翻译：

赖氨酸245在酿酒酵母中3-磷酸甘油脱氢酶（Gpd1）的稳定性和功能中起关键作用

甘油 3-磷酸脱氢酶 (Gpd1 异构体) 催化甘油合成的限速步骤，是酵母中渗透响应机制的关键组成部分。这种酶的三维结构类似于许多其他生物体的酶，包括人类。最近对人类酶的一项研究表明，K120（酵母中的 K152）处于正确的催化方向。K204（酵母中的 K245）不在平面上，不参与催化循环。目前的工作是为了确定 K245 在酵母 Gpd1 催化循环中的作用。发现 K245A 突变体具有较低的催化活性。与表现出约 60% 增加的野生型细胞相比，表达 Gpd1 (K245A) 的渗透胁迫细胞显示细胞内甘油没有变化。荧光显微镜，天然聚丙烯酰胺凝胶电泳 (PAGE) 分析、荧光光谱和硫黄素 T 荧光光谱法显示突变体的构象相对不稳定、易于聚集和降解。计算机研究显示 K245 周围有一个聚集“热点”。本研究确立了 K245 对 Gpd1 构象稳定性和功能适应的要求酿酒酵母。

更新日期：2021-08-08

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