Hydroxymethylbilane synthase

 

hydroxymethylbilane synthase (HemC) is involved in the tetrapolymerisation of porphobilinogen into the hydroxymethylbilane pre-uroporphyrinogen. It catalyses the reaction in a series of stepwise additions of pyrrolylmethyl groups until a hexapyrrole is present at the active centre. The terminal tetrapyrrole is then hydrolysed to yield the product, leaving a cysteine-bound dipyrrole on which assembly continues.

 

Reference Protein and Structure

Sequence
P06983 UniProt (2.5.1.61) IPR000860 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1gtk - Time-resolved and static-ensemble structural chemistry of hydroxymethylbilane synthase (1.66 Å) PDBe PDBsum 1gtk
Catalytic CATH Domains
3.30.160.40 CATHdb 3.40.190.10 CATHdb (see all for 1gtk)
Cofactors
Dipyrromethane cofactor(4-) (1)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.61)

water
CHEBI:15377ChEBI
+
porphobilinogen(1-)
CHEBI:58126ChEBI
→
preuroporphyrinogen(8-)
CHEBI:57845ChEBI
+
ammonium
CHEBI:28938ChEBI
Alternative enzyme names: HMB-synthase, Porphobilinogen ammonia-lyase (polymerizing), Porphobilinogen deaminase, Pre-uroporphyrinogen synthase, Uroporphyrinogen I synthase, Uroporphyrinogen I synthetase, Uroporphyrinogen synthase, Uroporphyrinogen synthetase, (4-(2-carboxyethyl)-3-(carboxymethyl)pyrrol-2-yl)methyltransferase (hydrolyzing),

Enzyme Mechanism

Introduction

The enzyme works by stepwise addition of pyrrolylmethyl groups until a hexapyrrole is present at the active centre. A single cycle involved the abstraction of a proton from porphobilinogen substrate by Asp84 to eliminate ammonia and produce the reactive azafulvene intermediate. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit. This occurs a further three times. The terminal tetrapyrrole is then hydrolysed to yield the product, leaving a cysteine-bound dipyrrole on which assembly continues.

Catalytic Residues Roles

UniProt PDB* (1gtk)
Cys242 Cys242A This is the residue in the active site to which the cofactor is covalently attached. It is currently still unclear how the cysteine-bound dipyrrole is formed. There are two current alternative mechanisms that have been suggested. Either by a stepwise addition of porphobilinogen to the cysteine [PMID:1522882, PMID:8117733], or by addition of the preuroporphyinogen and thus the first catalytic turnover of the enzyme proceeds via the addition of only a further two porphobilinogen molecules [PMID:8687374]. covalently attached
Asp84 Asp84A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Arg131, Arg132, Lys83, Arg149, Arg155 Arg131A, Arg132A, Lys83A, Arg149A, Arg155A Act to stabilise and activate the substrates and cofactor. activator, hydrogen bond donor, electrostatic stabiliser
*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

aromatic bimolecular elimination, proton transfer, overall reactant used, overall product formed, intermediate formation, deamination, proton relay, aromatic bimolecular nucleophilic addition, cofactor used, aromatic bimolecular nucleophilic substitution, native state of cofactor regenerated, intermediate terminated, native state of enzyme regenerated

References

  1. Woodcock SC et al. (1994), Biochemistry, 33, 2688-2695. Evidence for participation of aspartate-84 as a catalytic group at the active site of porphobilinogen deaminase obtained by site-directed mutagenesis of the hemC gene from Escherichia coli. DOI:10.1021/bi00175a043. PMID:8117733.
  2. Bung N et al. (2014), PLoS Comput Biol, 10, e1003484-. Structural insights into E. coli porphobilinogen deaminase during synthesis and exit of 1-hydroxymethylbilane. DOI:10.1371/journal.pcbi.1003484. PMID:24603363.
  3. Roberts A et al. (2013), Acta Crystallogr D Biol Crystallogr, 69, 471-485. Insights into the mechanism of pyrrole polymerization catalysed by porphobilinogen deaminase: high-resolution X-ray studies of the Arabidopsis thaliana enzyme. DOI:10.1107/S0907444912052134. PMID:23519422.
  4. Shoolingin-Jordan PM et al. (1996), Biochem J, 316, 373-376. Discovery that the assembly of the dipyrromethane cofactor of porphobilinogen deaminase holoenzyme proceeds initially by the reaction of preuroporphyrinogen with the apoenzyme. DOI:10.1042/bj3160373. PMID:8687374.
  5. Louie GV et al. (1992), Nature, 359, 33-39. Structure of porphobilinogen deaminase reveals a flexible multidomain polymerase with a single catalytic site. DOI:10.1038/359033a0. PMID:1522882.

Step 1. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Asp84A proton donor, proton acceptor

Chemical Components

ingold: aromatic bimolecular elimination, proton transfer, overall reactant used, overall product formed, intermediate formation, deamination, proton relay

Step 2. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Asp84A proton acceptor, proton donor

Chemical Components

proton transfer, ingold: aromatic bimolecular nucleophilic addition, cofactor used, intermediate formation, proton relay

Step 3. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Asp84A proton donor, proton acceptor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 4. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg149A activator
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A electrostatic stabiliser
Asp84A proton donor, proton acceptor

Chemical Components

ingold: aromatic bimolecular elimination, proton transfer, overall reactant used, overall product formed, intermediate formation, deamination, proton relay

Step 5. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A activator, electrostatic stabiliser
Asp84A proton acceptor, proton donor

Chemical Components

proton transfer, ingold: aromatic bimolecular nucleophilic addition, cofactor used, intermediate formation, proton relay

Step 6. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A activator
Arg149A electrostatic stabiliser
Asp84A proton acceptor, proton donor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 7. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg149A activator
Arg155A activator
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Asp84A proton acceptor, proton donor

Chemical Components

ingold: aromatic bimolecular elimination, proton transfer, overall reactant used, overall product formed, intermediate formation, deamination, proton relay

Step 8. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A activator
Arg155A activator
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Asp84A proton acceptor, proton donor

Chemical Components

proton transfer, ingold: aromatic bimolecular nucleophilic addition, cofactor used, intermediate formation, proton relay

Step 9. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A activator
Arg155A activator
Asp84A proton acceptor, proton donor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 10. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Arg149A activator
Arg155A activator
Asp84A proton acceptor, proton donor

Chemical Components

ingold: aromatic bimolecular elimination, proton transfer, overall reactant used, overall product formed, intermediate formation, deamination, proton relay

Step 11. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Arg149A activator
Arg155A activator
Asp84A proton donor, proton acceptor

Chemical Components

proton transfer, ingold: aromatic bimolecular nucleophilic addition, cofactor used, intermediate formation, proton relay

Step 12. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg149A activator
Arg155A activator
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor, proton relay
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Asp84A proton donor, proton acceptor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 13. Asp84 deprotonates N of the second pyrrole in the chain, causing double bond rearrangement to protonate the chain at the C4 position, this proton comes from a bound water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A activator
Arg155A activator
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Asp84A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 14. The hydroxide initiates a nucleophilic attack on the intermediate in an substitution reaction. The cofactor is re-protonated from Asp84

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys83A hydrogen bond donor, electrostatic stabiliser, activator
Asp84A hydrogen bond acceptor, hydrogen bond donor
Arg131A hydrogen bond donor, electrostatic stabiliser, activator
Arg132A hydrogen bond donor, electrostatic stabiliser, activator
Cys242A covalently attached
Arg149A electrostatic stabiliser
Arg155A electrostatic stabiliser
Arg149A activator
Arg155A activator
Asp84A proton donor

Chemical Components

ingold: aromatic bimolecular nucleophilic substitution, proton transfer, native state of cofactor regenerated, intermediate terminated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Step 2. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Step 3. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Step 4. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Step 5. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Step 6. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Step 7. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Step 8. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Step 9. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Step 10. Asp84 deprotonates the porphobilinogen substrate, which causes rearrangement of the double bonds to eliminate ammonia and form the reactive azafulvene intermediate.

Step 11. Asp84 deprotonates the dipyrromethane cofactor, which causes rearrangement of the double bonds an addition to the azafulvene intermediate in a nucleophilic manner.

Step 12. Asp84 deprotonates C4 of the intermediate, causing double bond rearrangement to produce the cofactor elongated by one pyrrole unit.

Step 13. Asp84 deprotonates N of the second pyrrole in the chain, causing double bond rearrangement to protonate the chain at the C4 position, this proton comes from a bound water molecule.

Step 14. The hydroxide initiates a nucleophilic attack on the intermediate in an substitution reaction. The cofactor is re-protonated from Asp84

Products.

Contributors

Gemma L. Holliday, Craig Porter, Charity Hornby