title: Functional divergence and catalytic properties of dehydroascorbate reductase family proteins from Populus tomentosa.
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.
authors: College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.
authors: Zhen-Xin
authors: ZX
authors: Tang
authors: Hai-Ling
authors: HL
authors: Yang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032107:Populus / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D017422:Sequence Analysis, DNA
chemical_list: D010940:Plant Proteins; D049452:Green Fluorescent Proteins; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate)
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