Biochemical Properties and Physiological Roles of NADP-Dependent Malic Enzyme in Escherichia coli
2011
Wang, Baojuan, Anhui Normal University, Wuhu, P. R. China | Wang, Peng, Anhui Normal University, Wuhu, P. R. China | Zheng, Enxia, Anhui Normal University, Wuhu, P. R. China | Chen, Xiangxian, Anhui Normal University, Wuhu, P. R. China | Zhao, Hanjun, The Clinical Laboratory of Wuhu Second People's Hospital, Wuhu, P. R. China | Song, Ping, Anhui Normal University, Wuhu, P. R. China | Su, Ruirui, Anhui Normal University, Wuhu, P. R. China | Li, Xiaoning, Yijishan Hospital of Wannan Medical College, Wuhu, P. R. China | Zhu, Guoping, Anhui Normal University, Wuhu, P. R. China
Malic enzymes catalyze the reversible oxidative decarboxylation of L-malate using NAD(P)+ as a cofactor. NADP-dependent malic enzyme (MaeB) from Escherichia coli MG1655 was expressed and purified as a fusion protein. The molecular weight of MaeB was about 83 kDa, as determined by SDS-PAGE. The recombinant. MaeB showed a maximum activity at pH 7.8 and 46℃. MaeB activity was dependent on the presence of Mn²+ but was strongly inhibited by Zn²+. In order to understand the physiological roles, recombinant E. coli strains (icd∨NADP/ΔmaeB and icd∨NAD/ΔmaeB) containing NADP-dependent isocitrate dehydrogenase (IDH), or engineered NAD-dependent IDH with the deletion of the maeB gene, were constructed using homologous recombination. During growth on acetate, icd∨NAD/ΔmaeB grew poorly, having a growth rate only 60% that of the wild-type strain (icd∨NADP). Furthermore, icd∨NADP/ΔmaeB exhibited a 2-fold greater adaptability to acetate than icd∨NAD/ΔmaeB, which may be explained by more NADPH production for biosynthesis in icd∨NADP/ΔmaeB due to its NADP-dependent IDH. These results indicated that MaeB was important for NADPH production for bacterial growth on acetate. We also observed that MaeB activity was significantly enhanced (7.83-fold) in icd∨NAD, which was about 3-fold higher than that in icd∨NADP, when switching from glucose to acetate. The marked increase of MaeB activity was probably induced by the shortage of NADPH in icd∨NAD. Evidently, MaeB contributed to the NADPH generation needed for bacterial growth on two carbon compounds.
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