de Graef M R, Alexeeva S, Snoep J L, Teixeira de Mattos M J
Department of Microbiology, E. C. Slater Institute, BioCentrum Amsterdam, University of Amsterdam, 1018 WS Amsterdam, The Netherlands.
J Bacteriol. 1999 Apr;181(8):2351-7. doi: 10.1128/JB.181.8.2351-2357.1999.
Escherichia coli MC4100 was grown in anaerobic glucose-limited chemostat cultures, either in the presence of an electron acceptor (fumarate, nitrate, or oxygen) or fully fermentatively. The steady-state NADH/NAD ratio depended on the nature of the electron acceptor. Anaerobically, the ratio was highest, and it decreased progressively with increasing midpoint potential of the electron acceptor. Similarly, decreasing the dissolved oxygen tension resulted in an increased NADH/NAD ratio. As pyruvate catabolism is a major switch point between fermentative and respiratory behavior, the fluxes through the different pyruvate-consuming enzymes were calculated. Although pyruvate formate lyase (PFL) is inactivated by oxygen, it was inferred that the in vivo activity of the enzyme occurred at low dissolved oxygen tensions (DOT </= 1%). A simultaneous flux from pyruvate through both PFL and the pyruvate dehydrogenase complex (PDHc) was observed. In anaerobic cultures with fumarate or nitrate as an electron acceptor, a significant flux through the PDHc was calculated on the basis of the redox balance, the measured products, and the known biochemistry. This result calls into question the common assumption that the complex cannot be active under these conditions. In vitro activity measurements of PDHc showed that the cellular content of the enzyme varied with the internal redox state and revealed an activity for dissolved oxygen tension of below 1%. Whereas Western blots showed that the E3 subunit of PDHc (dihydrolipoamide dehydrogenase) did not vary to a large extent under the conditions tested, the E2 subunit (dihydrolipoamide acetyltransferase) amount followed the trend that was found for the in vitro PDHc activity. From this it is concluded that regulation of the PDHc is exerted at the E1/E2 operon (aceEF). We propose that the external redox state (measured as the midpoint potentials of those terminal acceptors with which the cell has sufficient capacity to react) is reflected by the internal redox state. The latter may subsequently govern both the expression and the activity of the two pyruvate-catabolizing enzymes.
大肠杆菌MC4100在厌氧葡萄糖限制恒化器培养物中生长，培养条件为存在电子受体（延胡索酸、硝酸盐或氧气）或完全发酵。稳态NADH/NAD比值取决于电子受体的性质。在厌氧条件下，该比值最高，并且随着电子受体中点电位的增加而逐渐降低。同样，降低溶解氧张力会导致NADH/NAD比值增加。由于丙酮酸分解代谢是发酵行为和呼吸行为之间的主要转换点，因此计算了通过不同丙酮酸消耗酶的通量。尽管丙酮酸甲酸裂解酶（PFL）会被氧气灭活，但据推测该酶在体内的活性发生在低溶解氧张力（DOT≤1%）时。观察到丙酮酸同时通过PFL和丙酮酸脱氢酶复合物（PDHc）。在以延胡索酸或硝酸盐作为电子受体的厌氧培养物中，根据氧化还原平衡、测量的产物和已知的生物化学知识计算出通过PDHc的显著通量。这一结果对该复合物在这些条件下不能发挥活性这一普遍假设提出了质疑。PDHc的体外活性测量表明，该酶的细胞含量随内部氧化还原状态而变化，并且显示出在溶解氧张力低于1%时有活性。而蛋白质免疫印迹显示，在测试条件下，PDHc的E3亚基（二氢硫辛酰胺脱氢酶）变化不大，E2亚基（二氢硫辛酰胺乙酰转移酶）的量则遵循体外PDHc活性的变化趋势。由此得出结论，PDHc的调节作用发生在E1/E2操纵子（aceEF）。我们提出，外部氧化还原状态（以细胞有足够反应能力的那些末端受体的中点电位来衡量）由内部氧化还原状态反映。后者随后可能会同时控制两种丙酮酸分解代谢酶的表达和活性。
