Glutamate-1-semialdehyde 2,1-aminomutase

 

Tetrapyrroles are large macrocyclic compounds derived from a common biosynthetic pathway. The end-product, uroporphyrinogen III, is used to synthesise a number of important molecules, including vitamin B12, haem, sirohaem, chlorophyll, coenzyme F430 and phytochromobilin.

This entry represents glutamate-1-semialdehyde (GSA) aminotransferase (EC:5.4.3.8), which catalyses a transamination reaction to produce 5-aminoaevulinic acid during the first stage of tetrapyrrole biosynthesis by the C5 pathway. It is a class III aminotransferase.

 

Reference Protein and Structure

Sequence
P24630 UniProt (5.4.3.8) IPR004639 (Sequence Homologues) (PDB Homologues)
Biological species
Synechococcus elongatus PCC 6301 (Bacteria) Uniprot
PDB
2gsa - CRYSTAL STRUCTURE OF GLUTAMATE-1-SEMIALDEHYDE AMINOMUTASE (AMINOTRANSFERASE, WILD-TYPE FORM) (2.4 Å) PDBe PDBsum 2gsa
Catalytic CATH Domains
3.40.640.10 CATHdb (see all for 2gsa)
Cofactors
Pyridoxal 5'-phosphate(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:5.4.3.8)

(S)-4-amino-5-oxopentanoic acid zwitterion
CHEBI:57501ChEBI
→
5-ammoniolevulinate
CHEBI:356416ChEBI
Alternative enzyme names: Glutamate-1-semialdehyde aminotransferase,

Enzyme Mechanism

Introduction

This is a pyridoxal-phosphate (PLP) dependent enzyme. It is possible that the same group of the enzyme is responsible for all the general acid and base steps of the mechanism. By analogy to aspartate aminotransferase this group is assumed to be the Lys273A [PMID:8519748,PMID:1730703].

Catalytic Residues Roles

UniProt PDB* (2gsa)
Lys273 Lys273(272)A Acts as a catalytic nucleophile (forming a covalent intermediate with the PLP cofactor). Also acts as a general acid/base. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge, electron pair acceptor, electron pair donor
Asp245 Asp245(244)A Acts to stabilise the PLP, enabling the cofactor to act as an electron sink. hydrogen bond acceptor, electrostatic stabiliser
Tyr150 Tyr150(149)A Holds the PLP cofactor in place (stops it rotating within the active site) to ensure the correct reaction occurs. steric role
*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

bimolecular nucleophilic addition, proton transfer, overall reactant used, cofactor used, intermediate formation, unimolecular elimination by the conjugate base, intermediate collapse, dehydration, schiff base formed, enzyme-substrate complex formation, enzyme-substrate complex cleavage, bimolecular elimination, intermediate terminated, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Pugh CE et al. (1992), J Biol Chem, 267, 1584-1588. Mechanism of glutamate semialdehyde aminotransferase. Roles of diamino- and dioxo-intermediates in the synthesis of aminolevulinate. PMID:1730703.
  2. Li S et al. (2018), Biochem Biophys Res Commun, 500, 804-809. Crystal structure of a glutamate-1-semialdehyde-aminomutase from Pseudomonas aeruginosa PAO1. DOI:10.1016/j.bbrc.2018.04.163. PMID:29684343.
  3. Hennig M et al. (1997), Proc Natl Acad Sci U S A, 94, 4866-4871. Crystal structure of glutamate-1-semialdehyde aminomutase: An  2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity. DOI:10.1073/pnas.94.10.4866. PMID:9144156.
  4. Brody S et al. (1995), Biochemistry, 34, 15918-15924. Characterization of the different spectral forms of glutamate 1-semialdehyde aminotransferase by mass spectrometry. DOI:10.1021/bi00049a006. PMID:8519748.
  5. Smith MA et al. (1992), Biochemistry, 31, 11249-11254. Glutamate 1-semialdehyde aminotransferase: anomalous enantiomeric reaction and enzyme mechanism. DOI:10.1021/bi00160a041. PMID:1445864.

Step 1. The amine of the PMP cofactor attacks the carbonyl carbon of the (S)-4-amino-5-oxopentanoate substrate in a nucleophilic addition. This results in a the oxyanion formed deprotonating the newly attached amine.

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Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Tyr150(149)A steric role

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used, cofactor used, intermediate formation

Step 2. The secondary amine that results from the initial attack initiates an elimination of water, resulting in the substrate being covalently bound as a Schiff base.

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Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A hydrogen bond donor
Tyr150(149)A steric role
Lys273(272)A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, intermediate formation, dehydration, schiff base formed

Step 3. The amine of Lys273 deprotonates the C1 of the PMP, which acts as an electron sink and undergoes a double bond rearrangement.

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Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A hydrogen bond acceptor
Tyr150(149)A steric role
Lys273(272)A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The PMP initiates a deprotonation of the Lys273 through its conjugated pi-bond system, resulting in the protonation at the covalently bound carbon of the intermediate.

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Catalytic Residues Roles

Residue Roles
Tyr150(149)A steric role
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A hydrogen bond donor
Lys273(272)A proton donor

Chemical Components

proton transfer, intermediate formation

Step 5. The amine of Lys273 attacks the PMP in a nucleophilic addition reaction, the secondary amine of the attached intermediate reprotonates from the bound Lys229

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Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Tyr150(149)A steric role
Lys273(272)A proton donor, nucleophile

Chemical Components

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

Step 6. The secondary amine that results from the initial attack initiates an elimination of the covalently bound PMP, and the free diamino intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A covalently attached
Tyr150(149)A steric role
Lys273(272)A electron pair donor

Chemical Components

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

Step 7. The original amine of the intermediate attacks the covalently bound PMP cofactor in a nucleophilic addition and the bound Lys273 deprotonates the newly attached amine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A covalently attached
Tyr150(149)A steric role
Lys273(272)A proton acceptor, electron pair acceptor

Chemical Components

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

Step 8. The secondary amine that results from the attack initiates an elimination of the covalently bound lysine, resulting in free PMP and lysine in a neutral state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Tyr150(149)A steric role
Lys273(272)A nucleofuge

Chemical Components

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

Step 9. Lys273 deprotonates the carbon next to the covalently attached nitrogen, which initiates a double bond rearrangement through the conjugated pi-bond system of PMP.

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Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A hydrogen bond acceptor
Tyr150(149)A steric role
Lys273(272)A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 10. The PMP initiates a deprotonation of the Lys273 through its conjugated pi-bond system, resulting in the protonation at the C1 of the PMP cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A hydrogen bond donor
Tyr150(149)A steric role
Lys273(272)A proton donor

Chemical Components

proton transfer, intermediate formation

Step 11. Water attacks the imine in a nucleophilic addition reaction, which deprotonates the attacking water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr150(149)A steric role
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation

Step 12. Lys273 deprotonates the hydroxyl group, initiating an elimination of the PMP cofactor and 5-aminolevulinate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp245(244)A hydrogen bond acceptor, electrostatic stabiliser
Lys273(272)A hydrogen bond acceptor
Tyr150(149)A steric role
Lys273(272)A proton acceptor

Chemical Components

ingold: bimolecular elimination, intermediate collapse, intermediate terminated, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The amine of the PMP cofactor attacks the carbonyl carbon of the (S)-4-amino-5-oxopentanoate substrate in a nucleophilic addition. This results in a the oxyanion formed deprotonating the newly attached amine.

Step 2. The secondary amine that results from the initial attack initiates an elimination of water, resulting in the substrate being covalently bound as a Schiff base.

Step 3. The amine of Lys273 deprotonates the C1 of the PMP, which acts as an electron sink and undergoes a double bond rearrangement.

Step 4. The PMP initiates a deprotonation of the Lys273 through its conjugated pi-bond system, resulting in the protonation at the covalently bound carbon of the intermediate.

Step 5. The amine of Lys273 attacks the PMP in a nucleophilic addition reaction, the secondary amine of the attached intermediate reprotonates from the bound Lys229

Step 6. The secondary amine that results from the initial attack initiates an elimination of the covalently bound PMP, and the free diamino intermediate.

Step 7. The original amine of the intermediate attacks the covalently bound PMP cofactor in a nucleophilic addition and the bound Lys273 deprotonates the newly attached amine.

Step 8. The secondary amine that results from the attack initiates an elimination of the covalently bound lysine, resulting in free PMP and lysine in a neutral state.

Step 9. Lys273 deprotonates the carbon next to the covalently attached nitrogen, which initiates a double bond rearrangement through the conjugated pi-bond system of PMP.

Step 10. The PMP initiates a deprotonation of the Lys273 through its conjugated pi-bond system, resulting in the protonation at the C1 of the PMP cofactor.

Step 11. Water attacks the imine in a nucleophilic addition reaction, which deprotonates the attacking water.

Step 12. Lys273 deprotonates the hydroxyl group, initiating an elimination of the PMP cofactor and 5-aminolevulinate product.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Carine Berezin, Craig Porter, James Willey