4a-hydroxytetrahydrobiopterin dehydratase

 

4a-Hydroxy-tetrahydrobiopterin dehydratase/DCoH is a bifunctional protein. In the cytoplasm it is an enzyme required for the regeneration of tetrahydrobiopterin, an essential cofactor for phenylalanine hydroxylase. In the nucleus it functions as a transcriptional coactivator by forming a 2:2 heterotetramer with the hepatic nuclear factor HNF1alpha (HNF1).

The enzymatic catalytic form is a tetramer whereas the mode of binding to the nuclear factor is as a heterotetramer. The enzyme catalyses the dehydration of 4a-carbinolamine biopterin cofactor, a substrate used by the aromatic acid hydroxylases and NO synthase. Mutations of DcoHin humans have been associated with elevated levels of phenylalanine and the excretion of large amounts of 7-substituted pterins.

 

Reference Protein and Structure

Sequence
P61459 UniProt (4.2.1.96) IPR001533 (Sequence Homologues) (PDB Homologues)
Biological species
Rattus norvegicus (Norway rat) Uniprot
PDB
1dco - DCOH, A BIFUNCTIONAL PROTEIN-BINDING TRANSCRIPTIONAL COACTIVATOR (2.3 Å) PDBe PDBsum 1dco
Catalytic CATH Domains
3.30.1360.20 CATHdb (see all for 1dco)
Click To Show Structure

Enzyme Reaction (EC:4.2.1.96)

4a-hydroxy-L-erythro-5,6,7,8-tetrahydrobiopterin
CHEBI:15642ChEBI
→
water
CHEBI:15377ChEBI
+
(6R)-6-(L-erythro-1,2-dihydroxypropyl)-7,8-dihydro-6H-pterin
CHEBI:15643ChEBI
Alternative enzyme names: 4-alpha-hydroxy-tetrahydropterin dehydratase, Pterin-4-alpha-carbinolamine dehydratase, Tetrahydrobiopterin dehydratase, 4a-hydroxytetrahydrobiopterin hydro-lyase,

Enzyme Mechanism

Introduction

His63, activated by Glu58 abstracts the N(8) proton. The anion resides on the C-OH group in an enolate intermediate. The elimination of water from C4a is stereospecific, with His62 and His80 acting as general acids towards the 4a(R) hydroxyl and 4a(S) hydroxyl respectively. The residues then abstract a proton from N(5), relieving the atoms positive charge. This leads to the collapse of the anionic tetrahedral intermediate which has been stabilised by the presence of an oxyanion hole, resulting in the regeneration of the carbonyl at C(4) and reprotonation of N(8) by His63.

Catalytic Residues Roles

UniProt PDB* (1dco)
Glu81 (main-N) Glu81A (main-N) The backbone amide forms an oxyanion hole, stabilising the oxyanion intermediate. hydrogen bond donor, electrostatic stabiliser
His80 His80A Acts as the general acid/base in the 4a(S),6(R) diastereoisomer. Putatively identified as a stabilising influence in the 4a(R),6(R) diastereoisomer reaction. electrostatic stabiliser
Glu58 Glu58A Glu58 activates His63 through a hydrogen bond to act as a general base towards the N(5) atom of the substrate. proton acceptor, hydrogen bond acceptor, electrostatic stabiliser, proton donor
His63 His63A The residue acts as a general base towards the N(5) atom of the substrate which leads to the formation of a tetrahedral oxyanion at C4. In the last step of the reaction, the residue reprotonates N(5), giving the quinoid dihydrobiopterin product. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Asp89 Asp89A Activates His62 to act as a general acid/base during the reaction. hydrogen bond acceptor, electrostatic stabiliser
His80 (main-N) His80A (main-N) The imidazole side chain acts as a general acid/base towards the 4a(S) stereoisomer of the substrate. The backbone amide is implicated in stabilising the oxyanion intermediate. hydrogen bond donor, electrostatic stabiliser
His62 His62A The imidazole side chain acts as a general acid/base towards the 4a(R) stereoisomer of the substrate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton 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, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, overall product formed, intermediate collapse, dehydration, intermediate terminated, native state of enzyme regenerated

References

  1. Rebrin I et al. (1998), Biochemistry, 37, 11246-11254. Stereospecificity and Catalytic Function of Histidine Residues in 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH†. DOI:10.1021/bi980663h. PMID:9698371.
  2. Hevel JM et al. (2008), Arch Biochem Biophys, 477, 356-362. Determinants of oligomerization of the bifunctional protein DCoHα and the effect on its enzymatic and transcriptional coactivator activities. DOI:10.1016/j.abb.2008.06.023. PMID:18644344.
  3. Cameron S et al. (2008), Mol Biochem Parasitol, 158, 131-138. Crystal structures of Toxoplasma gondii pterin-4a-carbinolamine dehydratase and comparisons with mammalian and parasite orthologues. DOI:10.1016/j.molbiopara.2007.12.002. PMID:18215430.
  4. Hevel JM et al. (2006), Mol Genet Metab, 88, 38-46. Can the DCoHα isozyme compensate in patients with 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH deficiency? DOI:10.1016/j.ymgme.2005.11.014. PMID:16423549.
  5. Köster S et al. (1998), Biol Chem, 379, 1427-1432. Pterin-4a-carbinolamine dehydratase from Pseudomonas aeruginosa: characterization, catalytic mechanism and comparison to the human enzyme. PMID:9894810.
  6. Cronk JD et al. (1996), Protein Sci, 5, 1963-1972. High-resolution structures of the bifunctional enzyme and transcriptional coactivator DCoH and its complex with a product analogue. DOI:10.1002/pro.5560051002. PMID:8897596.
  7. Koster S et al. (1996), Eur J Biochem, 241, 858-864. Location of the Active Site and Proposed Catalytic Mechanism of Pterin-4A-Carbinolamine Dehydratase. DOI:10.1111/j.1432-1033.1996.00858.x.
  8. Endrizzi J et al. (1995), Science, 268, 556-559. Crystal structure of DCoH, a bifunctional, protein-binding transcriptional coactivator. DOI:10.1126/science.7725101.
  9. Rebrin I et al. (1995), Biochemistry, 34, 5801-5810. Catalytic characterization of 4a-hydroxytetrahydropterin dehydratase. PMID:7727440.
  10. Citron BA et al. (1992), Proc Natl Acad Sci U S A, 89, 11891-11894. Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins. PMID:1465414.

Step 1. His63 deprotonates the substrate initiating a double bond rearrangement that forms an oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu58A hydrogen bond acceptor
His62A hydrogen bond donor
His63A hydrogen bond acceptor, hydrogen bond donor
His80A (main-N) hydrogen bond donor
Glu81A (main-N) hydrogen bond donor
Asp89A hydrogen bond acceptor, electrostatic stabiliser
His80A electrostatic stabiliser
His63A proton donor, proton acceptor
Glu58A proton acceptor
His63A proton relay

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. The substrate eliminates water, which obtains its proton from His62.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu58A hydrogen bond acceptor, electrostatic stabiliser
His62A hydrogen bond donor
His63A hydrogen bond donor
His80A (main-N) hydrogen bond donor, electrostatic stabiliser
Glu81A (main-N) hydrogen bond donor, electrostatic stabiliser
Asp89A hydrogen bond acceptor, electrostatic stabiliser
His80A electrostatic stabiliser
His62A proton donor

Chemical Components

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

Step 3. His62 deprotonates the substrate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu58A hydrogen bond acceptor, electrostatic stabiliser
His62A hydrogen bond acceptor, hydrogen bond donor
His63A hydrogen bond donor
His80A (main-N) hydrogen bond donor, electrostatic stabiliser
Glu81A (main-N) hydrogen bond donor, electrostatic stabiliser
Asp89A hydrogen bond acceptor
His80A electrostatic stabiliser
His62A proton acceptor

Chemical Components

proton transfer, intermediate formation

Step 4. The oxyanion collapses, initiating double bond rearrangement that results in the deprotonation of His63 and the formation of the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu58A hydrogen bond acceptor, electrostatic stabiliser
His62A hydrogen bond donor
His63A hydrogen bond donor
His80A (main-N) hydrogen bond donor, electrostatic stabiliser
Glu81A (main-N) hydrogen bond donor, electrostatic stabiliser
Asp89A hydrogen bond acceptor, electrostatic stabiliser
His63A proton acceptor
Glu58A proton donor
His63A proton donor, proton relay

Chemical Components

proton transfer, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His63 deprotonates the substrate initiating a double bond rearrangement that forms an oxyanion.

Step 2. The substrate eliminates water, which obtains its proton from His62.

Step 3. His62 deprotonates the substrate.

Step 4. The oxyanion collapses, initiating double bond rearrangement that results in the deprotonation of His63 and the formation of the final product.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, James W. Murray, Craig Porter