Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum
Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum
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DOI：
10.1099/00221287-143-4-1095
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发表时间：
1997-04-01
期刊：
影响因子：
2.8
通讯作者：
Sahm, H
中科院分区：
文献类型：
--
作者：
PetersWendisch, PG;Wendisch, VF;Sahm, H
最近发现，磷酸烯醇式丙酮酸羧基酶(PEPCx)对谷氨酸棒杆菌的生长和赖氨酸生产是必不可少的，这意味着该生物具有一种替代的抗溶性酶(S)。在谷氨酸杆菌的通透性细胞中，我们检测了丙酮酸羧基酶(PCX)的活性。低浓度的ADP、AMP和乙酰辅酶A可有效抑制该酶的活性。在乳酸或丙酮酸上生长的细胞PCX活性最高[45+/-5nmolmin(-1)(mg干重)-1]，而在葡萄糖或醋酸盐上生长的细胞PCX活性约低2~3倍，这表明PCX的形成受生长介质中碳源的调节。在低浓度生物素(<；5 mUg L(-1))下生长的细胞中，PCx活性急剧下降，表明该酶是一种生物素蛋白。谷氨酸杆菌野生型和PEPCx阴性突变株对葡萄糖的生长实验表明，突变体对生物素的需求显著高于野生型，而两株菌株对乳酸生长的生物素需求相同，对乙酸盐生长的生物素需求同样低。这些结果表明：(I)PCx是在没有PEPCx的情况下在葡萄糖上生长所必需的抗坏血酸酶，(Ii)PCx是即使在PEPCx存在的情况下在乳酸上生长所必需的抗坏血酶，以及(Iii)PCx对以醋酸盐为碳源的生长没有抗坏作用。为了支持这些结论，对不能在含有乳酸的最低限度的培养基上生长，但能够在含有葡萄糖或醋酸盐的培养基上生长的克隆进行筛选，从而分离出谷氨酸杆菌的PCX缺陷突变株。
The recent discovery that phosphoenolpyruvate carboxylase (PEPCx) is dispensable for growth and lysine production in Corynebacterium glutamicum implies that this organism possesses (an) alternative anaplerotic enzyme(s). In permeabilized cells of C. glutamicum, we detected pyruvate carboxylase (PCx) activity. This activity was effectively inhibited by low concentrations of ADP, AMP and acetyl-CoA. PCx activity was highest [45 +/- 5 nmol min(-1) (mg dry wt)(-1)] in cells grown on lactate or pyruvate, and was about two- to threefold lower when the cells were grown on glucose or acetate, suggesting that formation of PCx is regulated by the carbon source in the growth medium. In cells grown at low concentrations of biotin (< 5 mu g l(-1)), PCx activity was drastically reduced, indicating that the enzyme is a biotin protein. Growth experiments with the wild-type and a defined PEPCx-negative mutant of C. glutamicum on glucose showed that the mutant has a significantly higher demand for biotin than the wild-type, whereas both strains have the same high biotin requirement for growth on lactate and the same low biotin requirement for growth on acetate. These results indicate that (i) PCx is an essential anaplerotic enzyme for growth on glucose in the absence of PEPCx, (ii) PCx is an essential anaplerotic enzyme for growth on lactate even in the presence of PEPCx, and (iii) PCx has no anaplerotic significance for growth on acetate as the carbon source. In support of these conclusions, screening for clones unable to grow on a minimal medium containing lactate, but able to grow on a medium containing glucose or acetate, led to the isolation of PCx-defective mutants of C. glutamicum.