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Int. J. Mol. Sci. 2013, 14(7), 12994-13004; doi:10.3390/ijms140712994

Enzymatic Properties of Populus α- and β-NAD-ME Recombinant Proteins

1 State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China 2 College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China 3 Agricultural Botany Department, Faculty of Agriculture, Tanta University, Tanta 31527, Egypt These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 1 April 2013 / Revised: 11 June 2013 / Accepted: 14 June 2013 / Published: 24 June 2013
(This article belongs to the Section Biochemistry, Molecular Biology and Biophysics)
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Plant 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.
Keywords: NAD-malic enzyme; Populus trichocarpa; glutathione S-transferase fusion protein; enzymatic property NAD-malic enzyme; Populus trichocarpa; glutathione S-transferase fusion protein; enzymatic property
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.

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