Enzymatic characterization and crystal structure analysis of Chlamydomonas reinhardtii dehydroascorbate reductase and their implications for oxidative stress

Hsin Yang Chang*, Shu Tseng Lin, Tzu Ping Ko, Shu Mei Wu, Tsen Hung Lin, Yu Ching Chang, Kai Fa Huang, Tse Min Lee

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Dehydroascorbate reductase (DHAR) is a key enzyme for glutathione (GSH)-dependent reduction of dehydroascorbate (DHA) to recycled ascorbate (AsA) in plants, and plays a major role against the toxicity of reactive oxygen species (ROS). Previously, we proposed that the increase of AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity against photooxidative stress in Chlamydomonas reinhardtii. In the present work, we use site-directed mutagenesis and crystal structure analysis to elucidate the molecular basis of how C. reinhardtii DHAR (CrDHAR1) is involved in the detoxification mechanisms. Mutagenesis data show that the D21A, D21N and C22A mutations result in severe loss of the enzyme's function, suggesting crucial roles of Asp-21 and Cys-22 in substrate binding and catalysis. The mutant K11A also exhibits reduced redox activity (∼50%). The crystal structure of apo CrDHAR1 further provides insights into the proposed mechanism centering on the strictly conserved Cys-22, which is suggested to initiate the redox reactions of DHA and GSH. Furthermore, in vitro oxidation of the recombinant CrDHAR1 in the presence of 1 mM H2O2 has minor effects on the Km for the substrates but significantly reduces the kcat. The enzyme's activity and its mRNA abundance in the C. reinhardtii cells are increased by treatment with 0.2–1 mM H2O2 but decreased when H2O2 is ≥ 1.5 mM. The latter decrease is accompanied by oxidative damage and lower AsA concentrations. These biochemical and physiological data provide new insights into the catalytic mechanism of CrDHAR1, which protects the C. reinhardtii cells from oxidative stress-induced toxicity.

Original languageEnglish
Pages (from-to)144-155
Number of pages12
JournalPlant Physiology and Biochemistry
Volume120
DOIs
StatePublished - Nov 2017

Keywords

  • Chlamydomonas
  • Dehydroascorbate reductase
  • Enzyme kinetics
  • Oxidative stress
  • Site-directed mutagenesis
  • X-ray crystallography

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