1-aminocyclopropane-1-carboxylate deaminase

 

1-aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal phosphate-dependent enzyme which catalyses a cyclopropane ring-opening reaction, the irreversible conversion of ACC to ammonia and alpha-ketobutyrate. In plants, the latter is a precursor of the ripening hormone ethylene. Some plant growth-promoting rhizobacteria can produce 1-aminocyclopropane-1-carboxylate deaminase to enhance plant growth.

ACC deaminase is unique amongst PLP-dependent enzymes, since the ring cleavage catalysed by ACC deaminase cannot proceed through an α-carbanionic intermediate due to the lack of an abstractable alpha-hydrogen atom from the substrate ACC.

 

Reference Protein and Structure

Sequence
Q7M523 UniProt (3.5.99.7) IPR005965 (Sequence Homologues) (PDB Homologues)
Biological species
Cyberlindnera saturnus (Fungus) Uniprot
PDB
1f2d - 1-AMINOCYCLOPROPANE-1-CARBOXYLATE DEAMINASE (2.0 Å) PDBe PDBsum 1f2d
Catalytic CATH Domains
3.40.50.1100 CATHdb (see all for 1f2d)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:3.5.99.7)

1-aminocyclopropanecarboxylic acid zwitterion
CHEBI:58360ChEBI
+
water
CHEBI:15377ChEBI
→
2-oxobutanoate
CHEBI:16763ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: 1-aminocyclopropane-1-carboxylate endolyase (deaminating), ACC deaminase, 1-aminocyclopropane carboxylic acid deaminase,

Enzyme Mechanism

Introduction

The substrate ACC reacts with the PLP cofactor and with the enzyme ACC deaminase to first produce an internal aldimine between the PLP and the Lys residue of the protein. This is followed by an aminyl intermediate which produces the external aldimine. This is followed by nucleophilic attack by a basic residue on the pro-S beta-carbon of ACC. Ring opening is initiated, aided by a nearby second basic residue located on the protein which removes a proton from the pro-R beta-carbon. This results in the formation of a quinonoid. The quinonoid undergoes electronic rearrangement to form another quinonoid. This is followed by deprotonation of a nearby residue regaining its nucleophilic capacity (basic activity) on the protein backbone. This eventually produces an aminocrotonate and a quinoid. These products reversibly undergo hydrolysis to form α-ketobutyrate and ammonium, regenerating the internal aldimine.

Catalytic Residues Roles

UniProt PDB* (1f2d)
Tyr269 Tyr269A The residue is implicated in a proton relay from the surrounding solvent to the active site. covalently attached, nucleofuge, nucleophile, electrostatic stabiliser
Tyr295 Tyr295A The residue has been proposed to act as a nucleophile towards the pro-S carbon of the ACC substrate. The close interaction between the residue and the carboxylate group of ACC is thought to lower the pKa of the residue's hydroxyl group, increasing its nucleophilicity. In addition, Tyr 295 may interact with Tyr 268 to form a charge relay system that further enhances the reactivity of Tyr 295. proton acceptor, electrostatic stabiliser, proton donor
Lys51 Lys51A The residue acts as a nucleophile towards the PLP cofactor. The pKa of the residue is thought to be modified by the hydrophobic protein environment, making the residue less basic than in free solution. The residue is also thought to abstract the beta proton, forming the aldimine intermediate. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electron pair acceptor, electron pair donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex cleavage, unimolecular elimination by the conjugate base, schiff base formed, enzyme-substrate complex formation, decyclisation, bimolecular elimination, native state of cofactor regenerated, native state of enzyme regenerated, reaction occurs outside the enzyme, overall product formed, intramolecular elimination

References

  1. Hontzeas N et al. (2006), Biotechnol Adv, 24, 420-426. Reaction mechanisms of the bacterial enzyme 1-aminocyclopropane-1-carboxylate deaminase. DOI:10.1016/j.biotechadv.2006.01.006. PMID:16524684.
  2. Karthikeyan S et al. (2004), Biochemistry, 43, 13328-13339. Structural Analysis ofPseudomonas1-Aminocyclopropane-1-carboxylate Deaminase Complexes:  Insight into the Mechanism of a Unique Pyridoxal-5‘-phosphate Dependent Cyclopropane Ring-Opening Reaction†,‡. DOI:10.1021/bi048878g. PMID:15491139.
  3. Ose T et al. (2003), J Biol Chem, 278, 41069-41076. Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase: insight into PLP-dependent cyclopropane ring-opening reaction. DOI:10.1074/jbc.M305865200. PMID:12882962.

Step 1. The substrate initiates a nucleophilic attack on the covalently bound PLP-Lys Schiff base.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr295A electrostatic stabiliser
Tyr269A electrostatic stabiliser
Lys51A covalently attached
Lys51A proton acceptor, electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used

Step 2. Lys51 is eliminated from the PLP-cofactor to form the PLP-substrate Schiff base intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A electrostatic stabiliser
Tyr295A electrostatic stabiliser
Lys51A nucleofuge

Chemical Components

enzyme-substrate complex cleavage, ingold: unimolecular elimination by the conjugate base, schiff base formed

Step 3. Tyr269 initiates a nucleophilic attack on the propane ring of the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr295A electrostatic stabiliser
Tyr269A nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, decyclisation

Step 4. Tyr295 abstracts a proton from the intermediate, eliminating the Tyr269.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A covalently attached
Tyr295A proton acceptor
Tyr269A nucleofuge

Chemical Components

proton transfer, ingold: bimolecular elimination, enzyme-substrate complex cleavage

Step 5. The PLP cofactor undergoes rearrangement to abstract a proton from Tyr295.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A electrostatic stabiliser
Tyr295A proton donor

Chemical Components

proton transfer

Step 6. Lys51 initiates the nucleophilic attack that is the first step in the transaldimination reaction to release the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys51A proton donor, nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 7. In the final step of the transaldimination reaction, the PLP-Lys cofactor is regenerated and the enzyme's final product released.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys51A electron pair donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, native state of cofactor regenerated, native state of enzyme regenerated, schiff base formed

Step 8. The enzyme product undergoes hydrolysis to produce the final products. It is likely that this process occurs outside the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, ingold: bimolecular nucleophilic addition

Step 9. Final step of the external hydrolysis of the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

overall product formed, reaction occurs outside the enzyme, ingold: intramolecular elimination
Download: Image, Marvin File

Step 1. The substrate initiates a nucleophilic attack on the covalently bound PLP-Lys Schiff base.

Step 2. Lys51 is eliminated from the PLP-cofactor to form the PLP-substrate Schiff base intermediate.

Step 3. Tyr269 initiates a nucleophilic attack on the propane ring of the intermediate.

Step 4. Tyr295 abstracts a proton from the intermediate, eliminating the Tyr269.

Step 5. The PLP cofactor undergoes rearrangement to abstract a proton from Tyr295.

Step 6. Lys51 initiates the nucleophilic attack that is the first step in the transaldimination reaction to release the product.

Step 7. In the final step of the transaldimination reaction, the PLP-Lys cofactor is regenerated and the enzyme's final product released.

Step 8. The enzyme product undergoes hydrolysis to produce the final products. It is likely that this process occurs outside the active site.

Step 9. Final step of the external hydrolysis of the product.

Products.

Introduction

The substrate ACC reacts with the PLP cofactor and with the enzyme ACC deaminase to first produce an internal aldimine between the PLP and the Lys residue of the protein. This is followed by an aminyl intermediate which produces the external aldimine. Then there is an initial direct beta-proton abstraction, performed by a basic residue on the protein leading to a quinonoid. The quinonoid undergoes electronic rearrangement to form another quinonoid. This is followed by deprotonation of a nearby residue regaining its nucleophilic capacity (basic activity) on the protein backbone. This eventually produces an aminocrotonate and a quinoid. These products reversibly undergo hydrolysis to form alpha-ketobutyrate and ammonium, regenerating the internal aldimine.

Catalytic Residues Roles

UniProt PDB* (1f2d)
Tyr269 Tyr269A The residue is implicated in a proton relay from the surrounding solvent to the active site. electrostatic stabiliser
Tyr295 Tyr295A Tyr 295 may interact with Tyr 268 to form a charge relay system that further enhances the reactivity of the residues in the active site. proton acceptor, electrostatic stabiliser, proton donor
Lys51 Lys51A The residue acts as a nucleophile towards the PLP cofactor. The pKa of the residue is thought to be modified by the hydrophobic protein environment, making the residue less basic than in free solution. The residue is also thought to abstract the beta proton, forming the aldimine intermediate. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electron pair acceptor, electron pair donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, schiff base formed, enzyme-substrate complex cleavage, decyclisation, native state of cofactor regenerated, native state of enzyme regenerated, reaction occurs outside the enzyme, intramolecular elimination, overall product formed

References

  1. Hontzeas N et al. (2006), Biotechnol Adv, 24, 420-426. Reaction mechanisms of the bacterial enzyme 1-aminocyclopropane-1-carboxylate deaminase. DOI:10.1016/j.biotechadv.2006.01.006. PMID:16524684.
  2. Ose T et al. (2003), J Biol Chem, 278, 41069-41076. Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase: insight into PLP-dependent cyclopropane ring-opening reaction. DOI:10.1074/jbc.M305865200. PMID:12882962.

Step 1. The substrate initiates a nucleophilic attack on the covalently bound PLP-Lys Schiff base.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A electrostatic stabiliser
Tyr295A electrostatic stabiliser
Lys51A covalently attached
Lys51A proton acceptor, electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 2. Lys51 is eliminated from the PLP-cofactor to form the PLP-substrate Schiff base intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A electrostatic stabiliser
Tyr295A electrostatic stabiliser
Lys51A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, schiff base formed, enzyme-substrate complex cleavage

Step 3. Tyr295 abstracts a proton from the intermediate. The PLP cofactor acts as an electron sink to stabilise the reactive intermediate formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A electrostatic stabiliser
Tyr295A proton acceptor

Chemical Components

proton transfer, decyclisation

Step 4. The intermediate undergoes double bond rearrangement that results in the terminal ene group bring reduced.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A electrostatic stabiliser
Tyr295A proton donor

Chemical Components

proton transfer

Step 5. Lys51 initiates the nucleophilic attack that is the first step in the transaldimination reaction to release the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr269A electrostatic stabiliser
Lys51A proton donor, nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 6. In the final step of the transaldimination reaction, the PLP-Lys cofactor is regenerated and the enzyme's final product released.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys51A covalently attached, electron pair donor

Chemical Components

schiff base formed, ingold: unimolecular elimination by the conjugate base, native state of cofactor regenerated, native state of enzyme regenerated

Step 7. The enzyme product undergoes hydrolysis to produce the final products. It is likely that this process occurs outside the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, ingold: bimolecular nucleophilic addition, proton transfer

Step 8. Final step of the external hydrolysis of the product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

proton transfer, ingold: intramolecular elimination, overall product formed
Download: Image, Marvin File

Step 1. The substrate initiates a nucleophilic attack on the covalently bound PLP-Lys Schiff base.

Step 2. Lys51 is eliminated from the PLP-cofactor to form the PLP-substrate Schiff base intermediate.

Step 3. Tyr295 abstracts a proton from the intermediate. The PLP cofactor acts as an electron sink to stabilise the reactive intermediate formed.

Step 4. The intermediate undergoes double bond rearrangement that results in the terminal ene group bring reduced.

Step 5. Lys51 initiates the nucleophilic attack that is the first step in the transaldimination reaction to release the product.

Step 6. In the final step of the transaldimination reaction, the PLP-Lys cofactor is regenerated and the enzyme's final product released.

Step 7. The enzyme product undergoes hydrolysis to produce the final products. It is likely that this process occurs outside the active site.

Step 8. Final step of the external hydrolysis of the product.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday