Molecular characterization of a dehydroascorbate reductase from Pinus bungeana.
Abstract Dehydroascorbate reductase (DHAR) plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR (PbDHAR) from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcription-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coli following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity (19.84 micromol/min per mg) and high affinity (a K(m) of 0.08 mM) towards the substrates dehydroascorbate (DHA). Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55 degrees C. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.
Yang
Hai-Ling
HL
College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China.
Zhao
Ying-Ru
YR
Wang
Cai-Ling
CL
Yang
Zhi-Ling
ZL
Zeng
Qing-Yin
QY
Lu
Hai
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003683:Dehydroascorbic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D028223:Pinus / Q000201:enzymology* / Q000235:genetics*; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate); D005978:Glutathione; D003683:Dehydroascorbic Acid