Metabolic engineering of a non‐allosteric citrate synthase in an Escherichia coli citrate synthase mutant
Metabolic engineering of a non‐allosteric citrate synthase in an Escherichia coli citrate synthase mutant
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大肠杆菌柠檬酸合酶突变体中非变构柠檬酸合酶的代谢工程
DOI：
--
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发表时间：
1995
影响因子：
2.7
通讯作者：
C. Evans
中科院分区：
文献类型：
--
作者：
C. Evans
本研究通过代谢工程和高分辨率13 C NMR检查了Krebs三羧酸（TCA）循环的组织。在用变构大肠杆菌转化的大肠杆菌野生型（WT）和柠檬酸合酶突变株（CS-）中，测量了[1，2，3 - 13 C]丙酸盐通过TCA循环氧化为谷氨酸盐。大肠杆菌柠檬酸合酶（ECCS）或非变构猪柠檬酸合酶（PCS）。ECCS和PCS中谷氨酸C-2、C-3和C-4的13 C富集分数相似;尽管在ECCS和PCS中观察到谷氨酸的总柠檬酸合酶活性和总C-4标记的定量差异。变构ECCS细胞的总酶活性比PCS低10倍，但谷氨酸C-4的总标记仅低50%，倍增时间相等。使用迭代程序（TCACALC）对观察到的光谱进行数学拟合，并得出ECCS和PCS的乙酸盐/琥珀酰辅酶A通量比均为10，该结果与[3 - 13 C]丙酸盐或[2 - 13 C]丙酸盐呈现的细胞的13 C光谱的同位素异构体分析一致。结果与ECCS中存在变构柠檬酸合酶和PCS中存在非变构柠檬酸合酶一致。前者通过由正变构机制激活的替代丙酸途径维持TCA循环通量，后者通过升高的酶水平维持TCA循环通量。
This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high‐resolution 13C NMR. The oxidation of [1,2,3‐13C]propionate to glutamate via the TCA cycle was measured in wild‐type (WT) and a citrate synthase mutant (CS−) strain of Escherichia coli transformed with allosteric E. coli citrate synthase (ECCS) or non‐allosteric pig citrate synthase (PCS). The 13C fractional enrichment in glutamate C‐2, C‐3, and C‐4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C‐4 labeling of glutamate were observed in ECCS and PCS. Allosteric ECCS cells contained 10‐fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C‐4 and equivalent doubling times. The observed spectra were mathematically fitted using an iterative procedure(TCACALC) and yielded an acetate/succinyl‐CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with [3‐13C] propionate or [2‐13C]propionate. The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non‐allosteric citrate synthase in PCS. The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels.
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酿酒酵母 TCA 循环中方向保守转移的证据：13C NMR 研究。
影响因子：
2.9
作者：
Sumegi,B;Sherry,AD;Malloy,CR;Srere,PA
通讯作者：
Srere,PA
DOI：
10.1016/s0021-9258(18)68590-4
发表时间：
1988-05
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Craig R. MalloyS;A. D. Sherrysll;Mark;Jeffrey
通讯作者：
Craig R. MalloyS;A. D. Sherrysll;Mark;Jeffrey
定点突变猪柠檬酸合酶的研究。
DOI：
10.1016/0006-291x(89)91831-7
发表时间：
1989
影响因子：
3.1
作者：
Evans,CT;Owens,D;Casazza,JP;Srere,PA
通讯作者：
Srere,PA
酿酒酵母中柠檬酸合酶的去抑制可能发生在转录水平。
影响因子：
5.3
作者：
Hoosein,MA;Lewin,AS
通讯作者：
Lewin,AS