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

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

1,2,†
,
1,†
,
3
 and
1,*
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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Abstract

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 3.0).

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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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