Aminodeoxychorismate lyase (class IV)

 

4-Amino-4-Deoxychorismate Lyase (ADCL), sourced from Escherichia coli is a member of the fold-type IV of PLP dependent enzymes. It converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate during the process of tetrahydrofolate biosynthesis. P-aminobenzoate is a precursor of folic acid. ADCL is a homodimer and shows the re-face specific hydrogen transfer typical of a member of the fold-type IV family.

 

Reference Protein and Structure

Sequence
P28305 UniProt (4.1.3.38) IPR017824 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1et0 - CRYSTAL STRUCTURE OF AMINODEOXYCHORISMATE LYASE FROM ESCHERICHIA COLI (2.2 Å) PDBe PDBsum 1et0
Catalytic CATH Domains
3.20.10.10 CATHdb 3.30.470.10 CATHdb (see all for 1et0)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:4.1.3.38)

4-amino-4-deoxychorismate(1-)
CHEBI:58406ChEBI
→
4-aminobenzoate
CHEBI:17836ChEBI
+
pyruvate
CHEBI:15361ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: 4-amino-4-deoxychorismate lyase, Enzyme X, ADC lyase, 4-amino-4-deoxychorismate pyruvate-lyase,

Enzyme Mechanism

Introduction

Lys159 forms a Schiff base linkage to PLP, attaching it to the active site. The amino group of ADC nucleophilically attacks the C4' of PLP. This forms a PLP-ADC Schiff base linkage and releases Lys159. Lys159 forms a hydrogen bond with Thr38, decreasing the free energy level of the external aldimine form of the enzyme.The hydroxyl of Thr38 makes a van der Waals contact with the methylene group of ADC. The alpha-carbon of the substrate is activated by the protonated Schiff base and the protonated pyridine ring. Lys159 acts as a general base and removes the alpha-proton of the alpha-carbon. This results in a quinonoid intermediate. The quinonoid intermediate is stabilised by the delocalisation of the cofactor-substrate Pi-system and the hydrogen bond between N1-H of the cofactor ring and Glu193. Thr38 acts as a general acid and protonates the olefin moiety, causing electrons to migrate towards the substrate from the cofactor. This results in release of pyruvate with aromatization of the six-membered ring of the substrate. Lys 159 nucleophilically attacks the C4' of PLP. This forms a PLP-Lys 159 Schiff base linkage and releases the substrate.

Catalytic Residues Roles

UniProt PDB* (1et0)
Lys140 Lys159(140)A Lys 159 forms a Schiff base linkage to PLP, attaching it to the active site. It also acts as a general base and removes the alpha-proton of the alpha-carbon. Lys 159 releases the substrate from the PLP-ADC Schiff base linkage at the end of the reaction. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge, electron pair acceptor, electron pair donor
Glu173 Glu193(173)A Glu 193 hydrogen bonds to the N1-H of the cofactor ring, helping to stabilise the quinonoid intermediate. hydrogen bond acceptor, electrostatic stabiliser
Thr28 Thr38(28)A Thr 38 acts as a general acid and protonates the olefin moiety. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
*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, intermediate formation, overall reactant used, enzyme-substrate complex formation, cofactor used, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, overall product formed, proton relay, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Nakai T et al. (2000), J Biochem, 128, 29-38. Three-Dimensional Structure of 4-Amino-4-Deoxychorismate Lyase from Escherichia coli. DOI:10.1093/oxfordjournals.jbchem.a022727. PMID:10876155.
  2. Dai YN et al. (2013), J Biol Chem, 288, 22985-22992. Structure and catalytic mechanism of yeast 4-amino-4-deoxychorismate lyase. DOI:10.1074/jbc.M113.480335. PMID:23818518.

Step 1. The amine of the substrate attacks the PLP cofactor in a nucleophilic addition and the bound Lys159 deprotonates the newly attached amine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A covalently attached, hydrogen bond acceptor, hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A proton acceptor, electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used, enzyme-substrate complex formation, cofactor used

Step 2. The secondary amine initiates an elimination of the covalently bound lysine, forming free PLP and lysine in a neutral state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A covalently attached, hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate formation, intermediate collapse, enzyme-substrate complex cleavage

Step 3. Lys159 deprotonates the CH adjacent to the bound amine, resulting in double bond rearrangement with the PLP cofactor acting as an electron sink.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A hydrogen bond acceptor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The PLP feeds the electrons back, resulting in the cleavage of the C-O attached to the aromatic ring and the C=C of the released substrate deprotonating Lys159.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor, proton relay
Lys159(140)A hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Thr38(28)A proton acceptor
Lys159(140)A proton donor
Thr38(28)A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate formation, overall product formed, proton relay, enzyme-substrate complex cleavage

Step 5. The amine of Lys159 attacks the PLP in a nucleophilic addition reaction, the secondary amine of the attached substrate reprotonates from the bound Lys159.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A nucleophile, proton donor

Chemical Components

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

Step 6. The secondary amine that results from the initial attack initiates an elimination of the covalently bound product, resulting in 4-aminobenzoate and the regenerated PLP cofactor

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A electron pair donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The amine of the substrate attacks the PLP cofactor in a nucleophilic addition and the bound Lys159 deprotonates the newly attached amine.

Step 2. The secondary amine initiates an elimination of the covalently bound lysine, forming free PLP and lysine in a neutral state.

Step 3. Lys159 deprotonates the CH adjacent to the bound amine, resulting in double bond rearrangement with the PLP cofactor acting as an electron sink.

Step 4. The PLP feeds the electrons back, resulting in the cleavage of the C-O attached to the aromatic ring and the C=C of the released substrate deprotonating Lys159.

Step 5. The amine of Lys159 attacks the PLP in a nucleophilic addition reaction, the secondary amine of the attached substrate reprotonates from the bound Lys159.

Step 6. The secondary amine that results from the initial attack initiates an elimination of the covalently bound product, resulting in 4-aminobenzoate and the regenerated PLP cofactor

Products.

Introduction

This alternative mechanism follows the same path as the other proposal other than pyruvate being eliminated intramolecularly. However the majority of evidence suggests the other mechanism is correct.

Catalytic Residues Roles

UniProt PDB* (1et0)
*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, intermediate formation, overall reactant used, enzyme-substrate complex formation, cofactor used, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, intramolecular elimination, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Culbertson JE et al. (2015), Biochemistry, 54, 2372-2384. Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes. DOI:10.1021/acs.biochem.5b00013. PMID:25710100.

Step 1. The amine of the substrate attacks the PLP cofactor in a nucleophilic addition and the bound Lys159 deprotonates the newly attached amine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A covalently attached, hydrogen bond acceptor, hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A proton acceptor, electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, overall reactant used, enzyme-substrate complex formation, cofactor used

Step 2. The secondary amine initiates an elimination of the covalently bound lysine, forming free PLP and lysine in a neutral state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A covalently attached, hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate formation, intermediate collapse, enzyme-substrate complex cleavage

Step 3. Pyruvate is eliminated intramolecularly, generating the aromatic ring.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu193(173)A electrostatic stabiliser
Thr38(28)A hydrogen bond acceptor
Glu193(173)A hydrogen bond acceptor
Thr38(28)A hydrogen bond donor
Lys159(140)A hydrogen bond donor

Chemical Components

ingold: intramolecular elimination, overall product formed

Step 4. The amine of Lys159 attacks the PLP in a nucleophilic addition reaction, the secondary amine of the attached substrate reprotonates from the bound Lys159.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A nucleophile, proton donor

Chemical Components

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

Step 5. The secondary amine that results from the initial attack initiates an elimination of the covalently bound product, resulting in 4-aminobenzoate and the regenerated PLP cofactor

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr38(28)A hydrogen bond donor, hydrogen bond acceptor
Lys159(140)A hydrogen bond donor
Glu193(173)A hydrogen bond acceptor, electrostatic stabiliser
Lys159(140)A electron pair donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, overall product formed, enzyme-substrate complex cleavage, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The amine of the substrate attacks the PLP cofactor in a nucleophilic addition and the bound Lys159 deprotonates the newly attached amine.

Step 2. The secondary amine initiates an elimination of the covalently bound lysine, forming free PLP and lysine in a neutral state.

Step 3. Pyruvate is eliminated intramolecularly, generating the aromatic ring.

Step 4. The amine of Lys159 attacks the PLP in a nucleophilic addition reaction, the secondary amine of the attached substrate reprotonates from the bound Lys159.

Step 5. The secondary amine that results from the initial attack initiates an elimination of the covalently bound product, resulting in 4-aminobenzoate and the regenerated PLP cofactor

Products.

Contributors

Gemma L. Holliday, Fiona J. E. Morgan, James Willey