十字花科黑腐病菌中葡萄糖-6-磷酸脱氢酶的酶学分析
发布时间：2019-07-12 08:25
【摘要】：葡萄糖-6-磷酸脱氢酶是磷酸戊糖途径(HMP)的限速酶,在十字花科黑腐病菌8004的基因组中,有2个基因XC1977和XC4082被注释为葡萄糖-6-磷酸脱氢酶(G6PD)。前期工作发现XC1977突变后,细胞的胞外多糖产量明显降低,而XC4082突变不影响胞外多糖产量。为明确这两个基因的编码产物在Xcc中的生化功能,本工作表达纯化了这两个蛋白,并对这两个蛋白的特征进行分析,发现这两个蛋白均具有6-葡萄糖脱氢酶的活性,但是它们的最适反应温度、最适反应pH、金属离子和烷化剂的敏感性完全不同。而且,XC1977以NAD~+和NADP~+为电子受体,而XC4082只能以NADP~+为电子受体。在以NAD~+和NADP~+为受体时,XC1977的K_m值分别为0.125 mmol/L和0.927 mmol/L,而XC4082以NADP~+为受体时的K_m值为0.364 mmol/L。这表明Xcc细胞合成胞外多糖时,需要NAD~+参与能量代谢。
[Abstract]:Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of pentose phosphate pathway (HMP). In the genome of Cruciferae black rot fungus 8004, two genes XC1977 and XC4082 are annotated as glucose-6-phosphate dehydrogenase (G6PD). It was found that the extracellular polysaccharide production decreased significantly after XC1977 mutation, but XC4082 mutation did not affect the extracellular polysaccharide yield. In order to clarify the biochemical function of the coding products of the two genes in Xcc, the two proteins were expressed and purified, and the characteristics of the two proteins were analyzed. It was found that the two proteins had the activity of 6-glucose dehydrogenase, but their optimum reaction temperature and sensitivity to pH, metal ions and alkylating agents were completely different. Moreover, XC1977 uses NAD~ and NADP~ as electron receptors, while XC4082 can only use NADP~ as electron receptors. When NAD~ and NADP~ were used as receptors, the km values of XC1977 were 0.125 mmol/L and 0.927 mmol/L, respectively, while the km values of XC4082 with NADP~ as receptors were 0.364 mmol/L.. This suggests that NAD~ is needed to participate in energy metabolism when Xcc cells synthesize extracellular polysaccharides.
【作者单位】： 广西农业科学院植物保护研究所;广西作物病虫害生物学重点实验室;广西大学生命科学与技术学院;广西大学亚热带农业生物资源保护利用国家重点实验室;
【基金】：国家自然科学基金项目(31660506) 广西自然科学基金(2015GXNSFBA139110) 广西农业科学院科技发展基金(桂农科2016JZ07) 广西科技计划项目(桂科AD16380018)共同资助
【分类号】：S432.42
本文编号：2513581
[Abstract]:Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of pentose phosphate pathway (HMP). In the genome of Cruciferae black rot fungus 8004, two genes XC1977 and XC4082 are annotated as glucose-6-phosphate dehydrogenase (G6PD). It was found that the extracellular polysaccharide production decreased significantly after XC1977 mutation, but XC4082 mutation did not affect the extracellular polysaccharide yield. In order to clarify the biochemical function of the coding products of the two genes in Xcc, the two proteins were expressed and purified, and the characteristics of the two proteins were analyzed. It was found that the two proteins had the activity of 6-glucose dehydrogenase, but their optimum reaction temperature and sensitivity to pH, metal ions and alkylating agents were completely different. Moreover, XC1977 uses NAD~ and NADP~ as electron receptors, while XC4082 can only use NADP~ as electron receptors. When NAD~ and NADP~ were used as receptors, the km values of XC1977 were 0.125 mmol/L and 0.927 mmol/L, respectively, while the km values of XC4082 with NADP~ as receptors were 0.364 mmol/L.. This suggests that NAD~ is needed to participate in energy metabolism when Xcc cells synthesize extracellular polysaccharides.
【作者单位】： 广西农业科学院植物保护研究所;广西作物病虫害生物学重点实验室;广西大学生命科学与技术学院;广西大学亚热带农业生物资源保护利用国家重点实验室;
【基金】：国家自然科学基金项目(31660506) 广西自然科学基金(2015GXNSFBA139110) 广西农业科学院科技发展基金(桂农科2016JZ07) 广西科技计划项目(桂科AD16380018)共同资助
【分类号】：S432.42
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