Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge?: Kinetics of NAD+ and NADH pools determined in vivo by 13C NMR
Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge?: Kinetics of NAD+ and NADH pools determined in vivo by 13C NMR
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DOI：
10.1074/jbc.m202573200
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发表时间：
2002-08-02
影响因子：
4.8
通讯作者：
Santos, H
中科院分区：
文献类型：
--
作者：
Neves, AR;Ventura, R;Santos, H
利用C-13和P-31 NMR技术，对乳酸乳球菌（Lactococcus lactis）糖酵解过程中NAD（+）、NADH、ATP、无机磷酸盐（Pi）、糖酵解中间产物和糖酵解终产物的动态变化进行了研究。合成了碳5上特异性标记的烟酸，并将其作为吡啶核苷酸的前体用于生长培养基中，以允许体内检测C-13标记的NAD（+）和NADH。L的容量。通过开启NADH氧化酶活性或通过敲除编码乳酸脱氢酶（LDH）的基因来操纵乳酸菌MG 1363再生NAD（+）。通过双交换构建LDH缺陷型菌株。在有氧和无氧条件下，在供糖条件下，亲本菌株（MG 1363）的NAD（+）是恒定的且最大（接近5 mm），而LDH-菌株的NAD（+）突然下降。只要葡萄糖可用，MG 1363中的NADH始终低于检测限。在LDH-菌株中，在厌氧条件下葡萄糖消耗速率低7倍，并且NADH达到高水平（2.5mM），反映了再生NAD（+）的严重限制。然而，在有氧条件下的糖酵解通量几乎一样高，在MG 1363尽管积累的NADH高达1.5毫米。甘油醛-3-磷酸脱氢酶能够支持高通量，即使在存在的NADH浓度远高于那些的亲本菌株。我们解释的数据表明，糖酵解通量野生型L。乳酸不是主要由NADH控制在甘油醛-3-磷酸甘油酸酯的水平。ATP/ADP/P-i含量可能起重要作用。
The involvement of nicotinamide adenine nucleotides (NAD(+), NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using C-13 and P-31 NMR to monitor in vivo the kinetics of the pools of NAD(+), NADH, ATP, inorganic phosphate (Pi), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of C-13-labeled NAD(+) and NADH. The capacity of L. lactis MG1363 to regenerate NAD(+) was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH- deficient strain was constructed by double crossover. Upon supply of glucose, NAD(+) was constant and maximal (similar to5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH- strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH- strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD(+). However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenate by NADH. The ATP/ADP/P-i content could play an important role.