State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, P.R. China.
BMC Biotechnol. 2024 Aug 22;24(1):58. doi: 10.1186/s12896-024-00884-z.
Based on our previous findings that salicylic acid and jasmonic acid increased Nostoc flagelliforme polysaccharide yield by regulating intracellular nitric oxide (NO) levels, the mechanism through which NO affects polysaccharide biosynthesis in Nostoc flagelliforme was explored from the perspective of S-nitrosylation (SNO). The addition of NO donor and scavenger showed that intracellular NO had a significant positive effect on the polysaccharide yield of N. flagelliforme. To explore the mechanism, we investigated the relationship between NO levels and the activity of several key enzymes involved in polysaccharide biosynthesis, including fructose 1,6-bisphosphate aldolase (FBA), glucokinase (GK), glucose 6-phosphate dehydrogenase (G6PDH), mitochondrial isocitrate dehydrogenase (ICDH), and UDP-glucose dehydrogenase (UGDH). The enzymatic activities of G6PDH, ICDH, and UGDH were shown to be significantly correlated with the shifts in intracellular NO levels. For further validation, G6PDH, ICDH, and UGDH were heterologously expressed in Escherichia coli and purified via Ni-NAT affinity chromatography, and subjected to a biotin switch assay and western blot analysis, which revealed that UGDH and G6PDH were susceptible to SNO. Furthermore, mass spectrometry analysis of proteins treated with S-nitrosoglutathione (GSNO) identified the SNO modification sites for UGDH and G6PDH as cysteine 423 and cysteine 249, respectively. These findings suggest that NO modulates polysaccharide biosynthesis in N. flagelliforme through SNO of UGDH and G6PDH. This reveals a potential mechanism through which NO promotes polysaccharide synthesis in N. flagelliforme, while also providing a new strategy for improving the industrial production of polysaccharides.
基于我们之前的发现，水杨酸和茉莉酸通过调节细胞内一氧化氮（NO）水平来提高发菜多糖的产量，本研究从 S-亚硝基化（SNO）的角度探讨了 NO 影响发菜多糖生物合成的机制。添加 NO 供体和清除剂表明，细胞内 NO 对发菜多糖产量有显著的正向影响。为了探讨这种机制，我们研究了 NO 水平与参与多糖生物合成的几种关键酶的活性之间的关系，包括 1,6-二磷酸果糖醛缩酶（FBA）、葡萄糖激酶（GK）、葡萄糖 6-磷酸脱氢酶（G6PDH）、线粒体异柠檬酸脱氢酶（ICDH）和 UDP-葡萄糖脱氢酶（UGDH）。结果表明，G6PDH、ICDH 和 UGDH 的酶活性与细胞内 NO 水平的变化显著相关。为了进一步验证，将 G6PDH、ICDH 和 UGDH 异源表达在大肠杆菌中，并通过 Ni-NAT 亲和层析进行纯化，然后进行生物素转换测定和 Western blot 分析，结果表明 UGDH 和 G6PDH 易发生 SNO。此外，用 S-亚硝基谷胱甘肽（GSNO）处理的蛋白质的质谱分析鉴定了 UGDH 和 G6PDH 的 SNO 修饰位点分别为半胱氨酸 423 和半胱氨酸 249。这些发现表明，NO 通过 UGDH 和 G6PDH 的 SNO 来调节发菜多糖的生物合成。这揭示了 NO 促进发菜多糖合成的潜在机制，同时也为提高多糖的工业生产提供了新策略。
