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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。
Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme in the pentose phosphate pathway (HMP). Two genes, XC1977 and XC4082, are annotated as glucose-6-phosphate dehydrogenase in the genome of C. cruciferica 8004 Enzyme (G6PD). Previous work found that XC1977 mutation, the cell yield of extracellular polysaccharide was significantly reduced, and XC4082 mutation does not affect the production of extracellular polysaccharide. In order to elucidate the biochemical functions of these two genes in Xcc, we expressed and purified these two proteins and analyzed the features of these two proteins. We found that both of these proteins have 6-glucose dehydrogenase However, their optimum reaction temperature, optimum reaction pH, and the sensitivity of metal ions and alkylating agents are completely different. Moreover, XC1977 with NAD ~ + and NADP ~ + as the electron acceptor, and XC4082 can only NADP ~ + as the electron acceptor. The K_m values of XC1977 were 0.125 mmol / L and 0.927 mmol / L for NAD ~ + and NADP ~ +, respectively, and 0.364 mmol / L for XC4082 with NADP ~ + as acceptor. This indicates that Xcc cells require extracellular polysaccharide NAD ~ + involved in energy metabolism.