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KMID : 0880220110490050797
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Journal of Microbiology
2011 Volume.49 No. 5 p.797 ~ p.802
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Biochemical properties and physiological roles of NADP-dependent malic enzyme in Escherichia coli
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Baojuan Wang
Peng Wang
Enxia Zheng
Xiangxian Chen
Ping Song
Ruirui Su
Guoping Zhu
Hanjun Zhao
Xiaoning Li
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Abstract
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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°C. 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 (icdNADP/ΔmaeB and icdNAD/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, icdNAD/ΔmaeB grew poorly, having a growth rate only 60% that of the wild-type strain (icdNADP). Furthermore, icdNADP/ΔmaeB exhibited a 2-fold greater adaptability to acetate than icdNAD/ΔmaeB, which may be explained by more NADPH production for biosynthesis in icdNADP/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 icdNAD, which was about 3-fold higher than that in icdNADP, when switching from glucose to acetate. The marked increase of MaeB activity was probably induced by the shortage of NADPH in icdNAD. Evidently, MaeB contributed to the NADPH generation needed for bacterial growth on two carbon compounds.
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KEYWORD
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NADP-dependent malic enzyme, biochemical property, homologous recombination, growth rate, NADPH
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