Literature summary for 4.2.1.1 extracted from

McGurn, L.D.; Moazami-Goudarzi, M.; White, S.A.; Suwal, T.; Brar, B.; Tang, J.Q.; Espie, G.S.; Kimber, M.S.
The structure, kinetics and interactions of the beta-carboxysomal beta-carbonic anhydrase, CcaA (2016), Biochem. J., 473, 4559-4572 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene ccmM, CcmM sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Synechocystis sp. PCC 6803
gene icfA, CcaA sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Synechocystis sp. PCC 6803 / Kazusa

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant truncated enzyme CcaA220, by sitting drop vapor diffusion method, mixing of 0.001 ml of 7 mg/ml protein solution with 0.001 ml of well solution containing 2.0 M Na formate and 0.1 M Na acetate, pH 4.6, X-ray diffraction structure determination and analysis at 1.45 A resolution, molecular replacement using the structure of a catalytic dimer (residues 121-318) from Pisum sativum beta-CA (PDB ID 1ekj) as a search model	Synechocystis sp. PCC 6803 / Kazusa

Crystallization (Comment)

Organism

purified recombinant truncated enzyme CcaA220, by sitting drop vapor diffusion method, mixing of 0.001 ml of 7 mg/ml protein solution with 0.001 ml of well solution containing 2.0 M Na formate and 0.1 M Na acetate, pH 4.6, X-ray diffraction structure determination and analysis at 1.45 A resolution, molecular replacement using the structure of a catalytic dimer (residues 121-318) from Pisum sativum beta-CA (PDB ID 1ekj) as a search model

Synechocystis sp. PCC 6803 / Kazusa

Inhibitors

Inhibitors	Comment	Organism	Structure
additional information	the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme	Synechocystis sp. PCC 6803 / Kazusa

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	stopped-flow spectrophotometric kinetic analysis	Synechocystis sp. PCC 6803 / Kazusa
additional information	-	additional information	stopped-flow spectrophotometric kinetic analysis	Synechocystis sp. PCC 6803

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
carboxysome	beta-carboxysome	Synechocystis sp. PCC 6803 / Kazusa	31470	-
carboxysome	beta-carboxysome	Synechocystis sp. PCC 6803	31470	-
additional information	CcaA's recruitment to the carboxysome relies upon interacting with CcmM by aligning their mutual three-fold symmetry axes	Synechocystis sp. PCC 6803 / Kazusa	-	-
additional information	CcaA's recruitment to the carboxysome relies upon interacting with CcmM by aligning their mutual three-fold symmetry axes	Synechocystis sp. PCC 6803	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Zn2+	required, metalloenzyme	Synechocystis sp. PCC 6803
Zn2+	required, metalloenzyme, the zinc ion bound to Cys39, His98, and Cys101 in the catalytic site, water forms the fourth zinc ligand	Synechocystis sp. PCC 6803 / Kazusa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
H2CO3	Synechocystis sp. PCC 6803 / Kazusa	-	CO2 + H2O	-	r
H2CO3	Synechocystis sp. PCC 6803	-	CO2 + H2O	-	r

Organism

Organism	UniProt	Comment	Textmining
Synechocystis sp. PCC 6803	P72758	-	-
Synechocystis sp. PCC 6803 / Kazusa	Q54735	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Synechocystis sp. PCC 6803 / Kazusa
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Synechocystis sp. PCC 6803

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
CO2 + H2O	-	Synechocystis sp. PCC 6803 / Kazusa	H2CO3	-	r
H2CO3	-	Synechocystis sp. PCC 6803 / Kazusa	CO2 + H2O	-	r
H2CO3	-	Synechocystis sp. PCC 6803	CO2 + H2O	-	r
additional information	CcaA-CcmM interactions, overview	Synechocystis sp. PCC 6803	?	-	?
additional information	protein/protein-binding analysis with recombinant enzyme variants CcaA220 and CcaA274 using surface plasmon resonance. CcaA-CcmM interactions, overview	Synechocystis sp. PCC 6803 / Kazusa	?	-	?

Subunits

Subunits	Comment	Organism
hexamer	enzyme CcaA shows a wellpacked trimer-of-dimers organization. The proximal part of the characteristic C-terminal extension is ordered by binding at a site that passes through the two-fold symmetry axis shared with an adjacent dimer, as a result, only one of a pair of converging termini can be ordered at any given time. beta-CA structures are organized around a tight, catalytic dimer. The catalytic site of CcaA is located at the interface between two protomers, with the zinc ion bound to Cys39, His98, and Cys101. A water molecule forms the fourth zinc ligand at pH 4.5	Synechocystis sp. PCC 6803 / Kazusa
More	CcmM is built as two distinct regions, the C-terminal region of CcmM consists of three to five repeats of a RubisCO small subunit-like domain, separated by flexible linker regions. This region binds RubisCO. The N-terminal domain is clearly homologous to gamma-CAs, but in at least some strains, including Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942, this domain lacks measurable CA activity. CcmM's N-terminal domain is trimeric. The C-terminal end of the eta-helix is dominated by the protruding alphaA helix and the alphaA-alphaB loop	Synechocystis sp. PCC 6803

Synonyms

Synonyms	Comment	Organism
beta carbonic anhydrase	-	Synechocystis sp. PCC 6803 / Kazusa
beta-CA	-	Synechocystis sp. PCC 6803 / Kazusa
beta-carbonic anhydrase	-	Synechocystis sp. PCC 6803 / Kazusa
CcaA	-	Synechocystis sp. PCC 6803 / Kazusa
CcmM	-	Synechocystis sp. PCC 6803
gamma-CA	-	Synechocystis sp. PCC 6803
gamma-carbonic anhydrase	-	Synechocystis sp. PCC 6803
icfA	-	Synechocystis sp. PCC 6803 / Kazusa

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Synechocystis sp. PCC 6803 / Kazusa
25	-	assay at	Synechocystis sp. PCC 6803

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
3340	-	CO2	recombinant full-length CcaA274 enzyme, pH 7.5, 25°C, CO2 hydration reaction	Synechocystis sp. PCC 6803 / Kazusa
13700	-	CO2	recombinant full-length CcaA274 enzyme, pH 9.5, 25°C, CO2 hydration reaction	Synechocystis sp. PCC 6803 / Kazusa
31000	-	CO2	recombinant truncated CcaA220 enzyme, pH 9.5, 25°C, CO2 hydration reaction	Synechocystis sp. PCC 6803 / Kazusa

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9.5	-	assay at, CO2 hydration reaction	Synechocystis sp. PCC 6803 / Kazusa

General Information

General Information	Comment	Organism
additional information	CcaA is crystallized at pH 4.5 but remains in the active, type I conformation, which is typified by a salt bridge between Asp41 and Arg43 that allows the zinc ion to remain free for substrate binding. In addition, CcaA does not show the co-operative inactivation at low pH characteristic of the type II bacterial enzymes (see below), and the residues that form the non-catalytic (regulatory) bicarbonate-binding site (Arg-Trp-Tyr) are absent. CcaA is a structurally characterized bacterial beta-CA that displays type I active sites	Synechocystis sp. PCC 6803 / Kazusa
additional information	homology modeling of Synechocystis CcmM	Synechocystis sp. PCC 6803
physiological function	CcaA is a beta-carbonic anhydrase that is a component of the carboxysomes of a subset of beta-cyanobacteria. It has a characteristic C-terminal extension and is recruited to the carboxysome via interactions with CcmM, UniProt ID P72758, which is itself a gamma-CA homologue with enzymatic activity in many, but not all cyanobacteria. Enzyme CcaA forms a complex with CcmM with sub-picomolar affinity, with contributions from residues in CcmM's alphaA helix and CcaA's C-terminal tail. Enzyme Cca shows low activity compared to other CAs, the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme. The need for the HCO3- entering the carboxysome to be efficiently converted into CO2 requires that carbonic anhydrase is co-encapsulated within the carboxysome. On the other hand, the presence of CA activity in the cytosol is highly deleterious as it converts the accumulated HCO3- into its membrane-permeable CO2 counterpart outside of the protective barrier afforded by the shell. Intracellular CAs in cyanobacteria are required to possess interaction determinants that target them to the carboxysome, as well as mechanisms that minimize CA activity prior to the completion of the shell. CcmM is the second potential beta-carboxysomal CA and is universally present in beta-carboxysomes as it functions as a central nexus for organizing the carboxysome's interior. Formation of the CcaA/CcmM complex probably requires significant backbone movements in at least one of the binding partners. CcaA is among the least active beta-CAs characterized to date, with activity comparable with the gamma-CA, CcmM	Synechocystis sp. PCC 6803 / Kazusa
physiological function	CcmM is itself a gamma-carbonic anhydrase homologue with enzymatic activity. It has a characteristic C-terminal extension and is recruited to the carboxysome via interactions with CcmM, which is itself a gamma-CA homologue with enzymatic activity in many, but not all cyanobacteria. Enzyme CcaA forms a complex with CcmM with sub-picomolar affinity, with contributions from residues in CcmM's alphaA helix and CcaA's C-terminal tail. CcaA is recruited to the beta-carboxysome by binding the N-terminal domain of CcmM. Enzyme Cca shows low activity compared to other CAs, the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme. The need for the HCO3- entering the carboxysome to be efficiently converted into CO2 requires that carbonic anhydrase is co-encapsulated within the carboxysome. On the other hand, the presence of CA activity in the cytosol is highly deleterious as it converts the accumulated HCO3- into its membrane-permeable CO2 counterpart outside of the protective barrier afforded by the shell. Intracellular CAs in cyanobacteria are required to possess interaction determinants that target them to the carboxysome, as well as mechanisms that minimize CA activity prior to the completion of the shell. CcmM is the second potential beta-carboxysomal CA and is universally present in beta-carboxysomes as it functions as a central nexus for organizing the carboxysome's interior. Formation of the CcaA/CcmM complex probably requires significant backbone movements in at least one of the binding partners. CcaA is among the least active beta-CAs characterized to date, with activity comparable with the gamma-CA, CcmM. CcmM is the second potential beta-carboxysomal CA and is universally present in beta-carboxysomes as it functions as a central nexus for organizing the carboxysome's interior. CcmM is built as two distinct regions, the C-terminal region of CcmM consists of three to five repeats of a RubisCO small subunit-like domain, separated by flexible linker regions, this region binds RubisCO. The N-terminal domain is clearly homologous to gamma-CAs, but in Synechocystis sp. PCC 6803, this domain lacks measurable CA activity	Synechocystis sp. PCC 6803

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
1190	-	CO2	recombinant full-length CcaA274 enzyme, pH 7.5, 25°C, CO2 hydration reaction	Synechocystis sp. PCC 6803 / Kazusa
17300	-	CO2	full-length CcaA274 enzyme, pH 9.5, 25°C, CO2 hydration reaction	Synechocystis sp. PCC 6803 / Kazusa