Literature summary for 4.2.1.1 extracted from

Benlloch, R.; Shevela, D.; Hainzl, T.; Grundstroem, C.; Shutova, T.; Messinger, J.; Samuelsson, G.; Sauer-Eriksson, A.E.
Crystal structure and functional characterization of photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii (2015), Plant Physiol., 167, 950-962 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3)pLysS	Chlamydomonas reinhardtii

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant detagged enzyme CrCAH3 with inhibitors phosphate and acetazolamide, hanging drop vapor diffusion method, mixing of 0.001 ml of 3.6 mg/l protein in mM Tris-HCl, pH 8.0, and 150 mM NaCl, and 1 mM ligand, with 0.001 ml of reservoir solution containing 2.5 M NH4H2PO4 and 0.1 M Tris-HCl, pH 8.0, to a final pH of pH 4.1, and equilibration over 1 ml reservoir solution, at 18°C, method optimization, X-ray diffraction structure determination and analysis at 2.6-2.7 A resolution	Chlamydomonas reinhardtii

Crystallization (Comment)

Organism

purified recombinant detagged enzyme CrCAH3 with inhibitors phosphate and acetazolamide, hanging drop vapor diffusion method, mixing of 0.001 ml of 3.6 mg/l protein in mM Tris-HCl, pH 8.0, and 150 mM NaCl, and 1 mM ligand, with 0.001 ml of reservoir solution containing 2.5 M NH4H2PO4 and 0.1 M Tris-HCl, pH 8.0, to a final pH of pH 4.1, and equilibration over 1 ml reservoir solution, at 18°C, method optimization, X-ray diffraction structure determination and analysis at 2.6-2.7 A resolution

Chlamydomonas reinhardtii

Protein Variants

Protein Variants	Comment	Organism
additional information	generation of a CrCIA3 deletion mutant. Point mutations A399C, C499G, C525G, A570G, G732C, G888C, and C903G, none of these mutations introduces changes in the CrCAH3 amino acid sequence	Chlamydomonas reinhardtii

Inhibitors

Inhibitors	Comment	Organism
acetazolamide	binding structure analysis, overview. Each unit cell is comprised of four dimers of CrCAH3	Chlamydomonas reinhardtii
DTT	50% inhibition at 0.5 mM, complete inhibition at above 2.5 mM	Chlamydomonas reinhardtii
phosphate	binding structure analysis, overview. Phosphate binds to the catalytic zinc cofactor, each unit cell is comprised of four dimers of CrCAH3	Chlamydomonas reinhardtii

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
additional information	enzyme CrCAH3 is associated with photosystem II, PSII	Chlamydomonas reinhardtii	-	-
thylakoid lumen	alpha-type CA (CrCAH3) locates in the thylakoid lumen	Chlamydomonas reinhardtii	31977	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Zn2+	required, metalloenzyme	Chlamydomonas reinhardtii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
H2CO3	Chlamydomonas reinhardtii	-	CO2 + H2O	-	r

Organism

Organism	UniProt	Comment	Textmining
Chlamydomonas reinhardtii	A8J4Z8	cell wall-deficient Chlamydomonas reinhardtii mutant92 (cw92), referred to as wild-type	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant is-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 (DE3)pLysS by nickel affinity chromatography, gel filtration, and tag cleavage by TEV protease, followed by dialysis, another step of nickel affinity chromatography, anion exchange chromatography, gel filtration, and ultrafiltration	Chlamydomonas reinhardtii

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
H2CO3	-	Chlamydomonas reinhardtii	CO2 + H2O	-	r

Subunits

Subunits	Comment	Organism
dimer	enzyme CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in alpha-type carbonic anhydrases	Chlamydomonas reinhardtii
More	each unit cell is comprised of four dimers of CrCAH3	Chlamydomonas reinhardtii

Synonyms

Synonyms	Comment	Organism
alpha-type CA	-	Chlamydomonas reinhardtii
CAH3	-	Chlamydomonas reinhardtii
CrCAH3	-	Chlamydomonas reinhardtii
luminal carbonic anhydrase	-	Chlamydomonas reinhardtii
photosystem II-associated carbonic anhydrase	-	Chlamydomonas reinhardtii

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
20	-	assay at	Chlamydomonas reinhardtii

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	6.5	-	Chlamydomonas reinhardtii

General Information

General Information	Comment	Organism
malfunction	a CrCIA3 deletion mutant shows 50% reduction of the O2 evolution rate	Chlamydomonas reinhardtii
additional information	a disulfide bond between Cys90 and Cys258 is essential for CrCAH3 activity	Chlamydomonas reinhardtii
physiological function	in the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3 improves proton removal from phorosystem PSII, possibly by rapid reformation of HCO3- from CO2. It plays a direct role of CrCAH3 in the turnover efficiency of PSII, and a stimulating effect of CrCAH3 and CO2/HCO3- on PSII activity. Possible redox regulation of the enzyme, overview. Redox regulation in the chloroplast thylakoid lumen is a common way to regulate lumenal and photosynthetic proteins	Chlamydomonas reinhardtii