Porphobilinogen synthase

 

5-Aminolaevulinic acid dehydratase (porphobilinogen synthase; EC 4.2.1.24) catalyses the dimerisation of two molecules of 5-aminolaevulinic acid to give porphobilinogen in a Knorr-type pyrrole synthesis. Porphobilinogen is the pyrrole precursor utilised by all living systems for the biosynthesis of tetrapyrroles, including haems, chlorophylls and corrins.

 

Reference Protein and Structure

Sequence
Q59643 UniProt (4.2.1.24) IPR001731 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas aeruginosa PAO1 (Bacteria) Uniprot
PDB
1gzg - Complex of a Mg2-dependent porphobilinogen synthase from Pseudomonas aeruginosa (mutant D139N) with 5-fluorolevulinic acid (1.66 Å) PDBe PDBsum 1gzg
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 1gzg)
Cofactors
Water (1), Sodium(+1) (1), Zinc(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:4.2.1.24)

5-ammoniolevulinate
CHEBI:356416ChEBI
→
water
CHEBI:15377ChEBI
+
porphobilinogen(1-)
CHEBI:58126ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: 5-levulinic acid dehydratase, Delta-aminolevulinate dehydratase, Delta-aminolevulinic acid dehydrase, Delta-aminolevulinic acid dehydratase, Delta-aminolevulinic dehydratase, Aminolevulinate dehydratase, Aminolevulinic dehydratase, 5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), HemB,

Enzyme Mechanism

Introduction

Two key lysine residues are located close to one another in the active and play a coordinated role in the mechanism of porphobilinogen synthesis, via formation of two Schiff bases with the two substrates. First P-side ALA binds and forms a Schiff base intermediate between C4 and one active site lysine (Lys260). This formally generates a water molecule, which is free to leave the open active site. A-side ALA binds next and forms a Schiff base intermediate between C4 and the adjacent active site lysine (Lys205). The next step in the reaction appears to be the formation of a bond between C3 of A-side ALA and C4 of P-side ALA, accompanied by the loss of one proton from C3 of A-side ALA. To complete formation of the pyrrole ring, the penultimate step in the reaction is a transfer of C4 of A-side ALA from its Schiff base with the active site lysine to a chemically comparable linkage with the C5-amino moiety of P-side ALA. This generates an almost-pyrrole intermediate. The final step in the PBGS catalysed reaction is the binding of P-side ALA to an adjacent active site of the octamer, which is then accompanied by conformational changes that open the neighbouring active site lid, which is accompanied by the breakdown of the covalent 'almost-product' complex and release of porphobilinogen. The residues performing general acid/base functions are yet to be formally identified.

Catalytic Residues Roles

UniProt PDB* (1gzg)
Lys260, Lys205 Lys260A, Lys205A Forms a Schiff base linkage with substrate via nucleophilic arttack on carbonyl group. Reaction proceeds as above. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleofuge, proton acceptor, polar interaction, proton donor, nucleophile, 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, dehydration, enzyme-substrate complex cleavage, schiff base formed, assisted tautomerisation (not keto-enol), intramolecular nucleophilic addition, cyclisation, bimolecular elimination, inferred reaction step, native state of enzyme regenerated

References

  1. Frère F et al. (2006), Biochemistry, 45, 8243-8253. Probing the Active Site ofPseudomonas aeruginosaPorphobilinogen Synthase Using Newly Developed Inhibitors†. DOI:10.1021/bi052611f. PMID:16819823.
  2. Mills-Davies N et al. (2017), Acta Crystallogr D Struct Biol, 73, 9-21. Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans, Escherichia coli and the hyperthermophile Pyrobaculum calidifontis . DOI:10.1107/s2059798316019525. PMID:28045381.
  3. Heinemann IU et al. (2010), Antimicrob Agents Chemother, 54, 267-272. Structure of the Heme Biosynthetic Pseudomonas aeruginosa Porphobilinogen Synthase in Complex with the Antibiotic Alaremycin. DOI:10.1128/aac.00553-09. PMID:19822707.
  4. Li N et al. (2009), Bioorg Chem, 37, 33-40. Probing the active site of rat porphobilinogen synthase using newly developed inhibitors. DOI:10.1016/j.bioorg.2008.11.001. PMID:19095280.
  5. Jaffe EK (2004), Bioorg Chem, 32, 316-325. The porphobilinogen synthase catalyzed reaction mechanism. DOI:10.1016/j.bioorg.2004.05.010. PMID:15381398.
  6. Frère F et al. (2002), J Mol Biol, 320, 237-247. Structure of Porphobilinogen Synthase from Pseudomonas aeruginosa in Complex with 5-Fluorolevulinic Acid Suggests a Double Schiff Base Mechanism. DOI:10.1016/s0022-2836(02)00472-2. PMID:12079382.
  7. Shoolingin-Jordan PM et al. (2002), Biochem Soc Trans, 30, 584-590. 5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism. DOI:10.1042/bst0300584. PMID:12196142.
  8. Erskine PT et al. (2001), J Mol Biol, 312, 133-141. The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors. DOI:10.1006/jmbi.2001.4947. PMID:11545591.
  9. Frankenberg N et al. (1999), J Mol Biol, 289, 591-602. High resolution crystal structure of a Mg2+-dependent porphobilinogen synthase. DOI:10.1006/jmbi.1999.2808. PMID:10356331.

Step 1. The zinc activated water deprotonates Lys260, activating it for the first step in Schiff base formation.

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Catalytic Residues Roles

Residue Roles
Lys260A hydrogen bond donor
Lys260A proton donor

Chemical Components

proton transfer

Step 2. Lys260 initiates a nucleophilic attack at the substrate's C4 carbonyl carbon in the first step of Schiff base formation.

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Catalytic Residues Roles

Residue Roles
Lys260A polar interaction, nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 3. In a proton rearrangement, the newly formed oxyanion deprotonates the positively charged nitrogen of Lys260.

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Catalytic Residues Roles

Residue Roles
Lys260A covalently attached, hydrogen bond donor, proton donor

Chemical Components

proton transfer

Step 4. The long pair of Lys260 initiates the elimination of the hydroxyl group as water, with the extra proton being donated by the zinc activated water.

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Catalytic Residues Roles

Residue Roles
Lys260A covalently attached, electron pair donor

Chemical Components

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

Step 5. The zinc activated water deprotonates Lys205, activating it for the first step in Schiff base formation.

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Catalytic Residues Roles

Residue Roles
Lys205A hydrogen bond donor
Lys260A covalently attached
Lys205A proton donor

Chemical Components

proton transfer

Step 6. Lys205 initiates a nucleophilic attack at the second substrate's C4 carbonyl carbon in the first step of Schiff base formation.

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Catalytic Residues Roles

Residue Roles
Lys260A covalently attached
Lys205A polar interaction, nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation

Step 7. In a proton rearrangement, the newly formed oxyanion deprotonates the positively charged nitrogen of Lys205.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys260A covalently attached
Lys205A covalently attached, hydrogen bond donor, proton donor

Chemical Components

proton transfer

Step 8. The long pair of Lys260 initiates the elimination of the hydroxyl group as water, with the extra proton being donated by the zinc activated water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys205A covalently attached
Lys260A covalently attached
Lys205A electron pair donor

Chemical Components

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

Step 9. Zinc activated water deprotonates the Lys205 bound intermediate at the C3 carbon, initiating a tautomerisation.

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Catalytic Residues Roles

Residue Roles
Lys260A covalently attached
Lys205A covalently attached
Lys205A electron pair acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol)

Step 10. The lone pair of Lys205 initiates a double bond rearrangement that results in a nucleophilic attack of the Lys205 bound intermediate upon the C4 carbonyl carbon of the Lys260 bound intermediate, forming the new C3-C4 double bond.

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Catalytic Residues Roles

Residue Roles
Lys205A covalently attached
Lys260A covalently attached
Lys205A electron pair donor
Lys260A electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition

Step 11. The amine group of the Lys260 bound intermediate initiates an intramolecular nucleophilic attack upon the C4 of the Lys205 bound intermediate, which in turn deprotonates the zinc activated water.

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Catalytic Residues Roles

Residue Roles
Lys260A covalently attached
Lys205A covalently attached, hydrogen bond acceptor
Lys205A proton acceptor, electron pair acceptor

Chemical Components

ingold: intramolecular nucleophilic addition, proton transfer, cyclisation

Step 12. Zinc activated water deprotonates the intermediate, causing an elimination of Lys205.

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Catalytic Residues Roles

Residue Roles
Lys260A covalently attached
Lys205A covalently attached
Lys205A nucleofuge

Chemical Components

ingold: bimolecular elimination, enzyme-substrate complex cleavage

Step 13. The covalently attached Lys260 deprotonates the zinc activated water.

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Catalytic Residues Roles

Residue Roles
Lys260A covalently attached, hydrogen bond acceptor, proton acceptor

Chemical Components

proton transfer

Step 14. Zinc activated water deprotonates the intermediate, causing an elimination of Lys260.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys260A covalently attached
Lys260A nucleofuge

Chemical Components

ingold: bimolecular elimination, enzyme-substrate complex cleavage

Step 15. Lys260 deprotonates the intermediate causing a double bond rearrangement that results in the final, aromatic, product.

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Catalytic Residues Roles

Residue Roles
Lys260A hydrogen bond acceptor, proton acceptor

Chemical Components

proton transfer

Step 16. Lys205 deprotonates the zinc activated water to regenerate the enzyme's starting state.

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Catalytic Residues Roles

Residue Roles
Lys205A hydrogen bond acceptor
Lys205A proton acceptor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The zinc activated water deprotonates Lys260, activating it for the first step in Schiff base formation.

Step 2. Lys260 initiates a nucleophilic attack at the substrate's C4 carbonyl carbon in the first step of Schiff base formation.

Step 3. In a proton rearrangement, the newly formed oxyanion deprotonates the positively charged nitrogen of Lys260.

Step 4. The long pair of Lys260 initiates the elimination of the hydroxyl group as water, with the extra proton being donated by the zinc activated water.

Step 5. The zinc activated water deprotonates Lys205, activating it for the first step in Schiff base formation.

Step 6. Lys205 initiates a nucleophilic attack at the second substrate's C4 carbonyl carbon in the first step of Schiff base formation.

Step 7. In a proton rearrangement, the newly formed oxyanion deprotonates the positively charged nitrogen of Lys205.

Step 8. The long pair of Lys260 initiates the elimination of the hydroxyl group as water, with the extra proton being donated by the zinc activated water.

Step 9. Zinc activated water deprotonates the Lys205 bound intermediate at the C3 carbon, initiating a tautomerisation.

Step 10. The lone pair of Lys205 initiates a double bond rearrangement that results in a nucleophilic attack of the Lys205 bound intermediate upon the C4 carbonyl carbon of the Lys260 bound intermediate, forming the new C3-C4 double bond.

Step 11. The amine group of the Lys260 bound intermediate initiates an intramolecular nucleophilic attack upon the C4 of the Lys205 bound intermediate, which in turn deprotonates the zinc activated water.

Step 12. Zinc activated water deprotonates the intermediate, causing an elimination of Lys205.

Step 13. The covalently attached Lys260 deprotonates the zinc activated water.

Step 14. Zinc activated water deprotonates the intermediate, causing an elimination of Lys260.

Step 15. Lys260 deprotonates the intermediate causing a double bond rearrangement that results in the final, aromatic, product.

Step 16. Lys205 deprotonates the zinc activated water to regenerate the enzyme's starting state.

Products.

Contributors

Gemma L. Holliday, Anna Waters, Craig Porter