Anderlund M, Nissen T L, Nielsen J, Villadsen J, Rydström J, Hahn-Hägerdal B, Kielland-Brandt M C
Department of Yeast Genetics, Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark.
Appl Environ Microbiol. 1999 Jun;65(6):2333-40. doi: 10.1128/AEM.65.6.2333-2340.1999.
We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.
我们研究了在表达来自大肠杆菌的膜结合转氢酶的重组酿酒酵母中，NAD(H)和NADP(H)辅酶系统相互转化的生理效应。我们的目的是确定膜结合转氢酶是否能在酿酒酵母中将NADH再氧化为NAD⁺，从而减少厌氧发酵过程中甘油的形成。从重组菌株中分离出的膜在有NADP⁺存在的情况下，表现出被NADPH和NADH还原3-乙酰吡啶-NAD⁺的能力，这表明存在活性酶。然而，与大肠杆菌的情况不同，大多数转氢酶活性并不存在于酵母质膜中；相反，该酶似乎仍定位于内质网膜。在厌氧葡萄糖发酵过程中，我们观察到在表达高水平转氢酶的菌株中，2-氧代戊二酸、甘油和乙酸的形成增加，这表明NADPH消耗增加和NADH产生增加。我们测量了表达转氢酶的细胞中NADH、NAD⁺、NADPH和NADP⁺的细胞内浓度。NADPH库的减少表明转氢酶将还原当量从NADPH转移到了NAD⁺。
