Literature summary for 1.8.5.1 extracted from

Chang, H.Y.; Lin, S.T.; Ko, T.P.; Wu, S.M.; Lin, T.H.; Chang, Y.C.; Huang, K.F.; Lee, T.M.
Enzymatic characterization and crystal structure analysis of Chlamydomonas reinhardtii dehydroascorbate reductase and their implications for oxidative stress (2017), Plant Physiol. Biochem., 120, 144-155 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene DHAR1, recombinant expression of wild-type and mutant enzymes in Escherichia coli	Chlamydomonas reinhardtii

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant full-length wild-type enzyme CrDHAR1 (amino acids 1-226), and mutants CrDHAR1DELTAN (amino acids 5-226) or CrDHAR1DELTAC (amino acids 1-218), sitting drop vapor diffusion method, mixing of protein solution with an equal volume of crystallization solution containing 0.1 M Tris-HCl, pH 8.0, and 2 M ammonium sulfate, 20°C, X-ray diffraction structure determination and analysis at 2.46-2.55 A resolution, molecular replacement method using the structure of reduced wild-type OsDHAR (5D9T) as search model, modeling	Chlamydomonas reinhardtii

Crystallization (Comment)

Organism

purified recombinant full-length wild-type enzyme CrDHAR1 (amino acids 1-226), and mutants CrDHAR1DELTAN (amino acids 5-226) or CrDHAR1DELTAC (amino acids 1-218), sitting drop vapor diffusion method, mixing of protein solution with an equal volume of crystallization solution containing 0.1 M Tris-HCl, pH 8.0, and 2 M ammonium sulfate, 20°C, X-ray diffraction structure determination and analysis at 2.46-2.55 A resolution, molecular replacement method using the structure of reduced wild-type OsDHAR (5D9T) as search model, modeling

Chlamydomonas reinhardtii

Protein Variants

Protein Variants	Comment	Organism
C22A	site-directed mutagenesis, the mutation results in severe loss of the enzyme's function	Chlamydomonas reinhardtii
D21A	site-directed mutagenesis, the mutation results in severe loss of the enzyme's function	Chlamydomonas reinhardtii
D21N	site-directed mutagenesis, the mutation results in severe loss of the enzyme's function	Chlamydomonas reinhardtii
K11A	site-directed mutagenesis, the mutant K11A exhibits about 50% reduced redox activity compared to wild-type	Chlamydomonas reinhardtii
additional information	generation of truncated mutants CrDHAR1DELTAN (amino acids 5-226) or CrDHAR1DELTAC (amino acids 1-218)	Chlamydomonas reinhardtii

Inhibitors

Inhibitors	Comment	Organism	Structure
H2O2	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 Chlamydomonas reinhardtii cells are increased by treatment with 0.2-1.0 mM H2O2 but decreased when H2O2 is over 1.5 mM	Chlamydomonas reinhardtii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.09	-	dehydroascorbate	pH 8.0, 22°C, recombinant wild-type enzyme	Chlamydomonas reinhardtii
0.27	-	glutathione	pH 8.0, 22°C, recombinant wild-type enzyme	Chlamydomonas reinhardtii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2 glutathione + dehydroascorbate	Chlamydomonas reinhardtii	-	glutathione disulfide + ascorbate	-	?

Organism

Organism	UniProt	Comment	Textmining
Chlamydomonas reinhardtii	A8I0K9	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli	Chlamydomonas reinhardtii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2 glutathione + dehydroascorbate	-	Chlamydomonas reinhardtii	glutathione disulfide + ascorbate	-	?

Synonyms

Synonyms	Comment	Organism
CrDHAR1	-	Chlamydomonas reinhardtii
dehydroascorbate reductase	-	Chlamydomonas reinhardtii
DHAR	-	Chlamydomonas reinhardtii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	-	Chlamydomonas reinhardtii

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
20	50	high activity range, overview	Chlamydomonas reinhardtii

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
390.06	-	glutathione	pH 8.0, 22°C, recombinant wild-type enzyme	Chlamydomonas reinhardtii
400.43	-	dehydroascorbate	pH 8.0, 22°C, recombinant wild-type enzyme	Chlamydomonas reinhardtii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	-	Chlamydomonas reinhardtii

pH Range

pH Minimum	pH Maximum	Comment	Organism
7	9	high activity range, overview	Chlamydomonas reinhardtii

Expression

Organism	Comment	Expression
Chlamydomonas reinhardtii	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 Chlamydomonas reinhardtii cells are increased by treatment with 0.2-1.0 mM H2O2 but decreased when H2O2 is over 1.5 mM	down

General Information

General Information	Comment	Organism
malfunction	the increase of AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity against photooxidative stress in Chlamydomonas reinhardtii	Chlamydomonas reinhardtii
additional information	the crystal structure of apo CrDHAR1 provides insights into the proposed mechanism centering on the strictly conserved Cys22, which is suggested to initiate the redox reactions of DHA and GSH, crucial roles of Asp21 and Cys22 in substrate binding and catalysis	Chlamydomonas reinhardtii
physiological function	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)	Chlamydomonas reinhardtii

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
1444.7	-	glutathione	pH 8.0, 22°C, recombinant wild-type enzyme	Chlamydomonas reinhardtii
4449.2	-	dehydroascorbate	pH 8.0, 22°C, recombinant wild-type enzyme	Chlamydomonas reinhardtii