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Molecular Biology Reports

, Volume 40, Issue 8, pp 5105–5114 | Cite as

Functional divergence and catalytic properties of dehydroascorbate reductase family proteins from Populus tomentosa

  • Zhen-Xin Tang
  • Hai-Ling Yang
Article

Abstract

Dehydroascorbate reductase (DHAR) is a key enzyme in the ascorbate–glutathione cycle that maintains reduced pools of ascorbic acid and serves as an important antioxidant. In this study, to investigate functional divergence of plant DHAR family and catalytic characteristics of the glutathione binding site (G-site) residues of DHAR proteins, we cloned three DHAR genes (PtoDHAR1/2/3) from Populus tomentosa and predicted the G-site residues. PtoDHAR1 protein was localized in chloroplast, while PtoDHAR2/3 proteins showed cytosolic localizations. Three DHAR proteins showed different enzymatic activities, apparent kinetic characteristics, optimum T m and pH profiles, indicating their functional divergence. Cys20, Lys8, Pro61, Asp72 and Ser73 of PtoDHAR2 were predicted as G-site residues based on their N-terminal amino acid sequence identity and the available crystal structures of glutathione S-transferases. The biochemical functions of these residues are examined in this study through site-directed mutagenesis. The aforementioned five residues are critical components of active sites that contribute to the enzyme’s catalytic activity. Cys20, Pro61 and Asp72 of PtoDHAR2 are also responsible for maintaining proper protein structure. This study provides new insights into the functional divergence of the plant DHAR family and biochemical properties of the G-site residues in plant DHAR proteins.

Keywords

Dehydroascorbate reductase Functional divergence Enzyme assay GSH-binding site Site-directed mutagenesis 

Notes

Acknowledgments

This study was supported by grants from the National Basic Research Program of China (2009CB119104) and Natural Science Foundation of China (NSFC 31270641).

Supplementary material

11033_2013_2612_MOESM1_ESM.doc (853 kb)
Supplementary material 1 (DOC 853 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.College of Life Sciences and BiotechnologyBeijing Forestry UniversityBeijingChina
  2. 2.The Key Laboratory of Tree and Ornamental Plant Breeding and Biotechnology of State Forestry AdministrationBeijingChina

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