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Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins
AbstractPlant mitochondrial NAD-malic enzyme (NAD-ME), which is composed of α- and β-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4) in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative α-subunits, while PtNAD-ME3 and -4 encode putative β-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that α-NAD-MEs are more active than β-NAD-MEs and that α- and β-NAD-MEs presented different kinetic properties (Vmax, kcat and kcat/Km). The effect of different amounts of metabolites on the activities of Populus α- and β-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all α- and β-NAD-MEs and glucose-6-P and fructose inhibited only the α-NAD-MEs.
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Liu, J.; Yu, Q.; Elsheery, N.I.; Cheng, Y. Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins. Int. J. Mol. Sci. 2013, 14, 12994-13004.View more citation formats
Liu J, Yu Q, Elsheery NI, Cheng Y. Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins. International Journal of Molecular Sciences. 2013; 14(7):12994-13004.Chicago/Turabian Style
Liu, Jinwen; Yu, Qiguo; Elsheery, Nabil I.; Cheng, Yuxiang. 2013. "Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins." Int. J. Mol. Sci. 14, no. 7: 12994-13004.