Dethiobiotin synthase

 

Dethiobiotin synthase is the penultimate enzyme in the biotin synthesis pathway in E. coli and other microorganisms. The enzyme catalyses the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanic acid (DAPA) and carbon dioxide. The enzyme requires ATP and divalent cations as cofactors. The enzyme represents a third enzymatic mechanism for carboxylation reactions, after the biotin-dependent carboxylases and ribulose-bisphosphate carboxylase.

 

Reference Protein and Structure

Sequence
P13000 UniProt (6.3.3.3) IPR004472 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1bs1 - DETHIOBIOTIN SYNTHETASE COMPLEXED WITH DETHIOBIOTIN, ADP , INORGANIC PHOSPHATE AND MAGNESIUM (1.8 Å) PDBe PDBsum 1bs1
Catalytic CATH Domains
3.40.50.300 CATHdb (see all for 1bs1)
Cofactors
Water (1), Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.3.3)

carbon dioxide
CHEBI:16526ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
7,8-diaminononanoate
CHEBI:17830ChEBI
→
hydron
CHEBI:15378ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
dethiobiotin(1-)
CHEBI:57861ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: Desthiobiotin synthase, DTB synthetase,

Enzyme Mechanism

Introduction

Detailed mechanistic information is sparse. However, speculations can be made on the basis of site-directed mutagenesis and crystal structures. The first step in the reaction is the formation of a carbamate of the substrate DAPA. Evidence is conflicting over whether this is on the N7 or N8 position of the substrate. The second step is the formation of a carbamic-phosphoric mixed anhydride as a second intermediate, followed by ring closure. This is most likely to occur via nucleophilic attack of the activated carbamate with a substrate nitrogen, with release of inorganic phosphate.

Catalytic Residues Roles

UniProt PDB* (1bs1)
Glu13 Glu12A Forms part of the magnesium 1 binding site. metal ligand
Thr12, Lys38, Lys16, Ser42 (main-N), Glu13, Ser42 Thr11A, Lys37A, Lys15A, Ser41A (main-N), Glu12A, Ser41A Helps to stabilise the reactive intermediates. electrostatic stabiliser
Asp55, Thr17, Glu116 Asp54A, Thr16A, Glu115A Forms part of the magnesium 2 binding site. metal ligand
*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, overall reactant used, intermediate formation, bimolecular nucleophilic substitution, overall product formed, intramolecular nucleophilic addition, cyclisation, unimolecular elimination by the conjugate base, dephosphorylation, intermediate terminated, intermediate collapse

References

  1. Yang G et al. (1997), Biochemistry, 36, 4751-4760. Active Site Mutants ofEscherichia coliDethiobiotin Synthetase:  Effects of Mutations on Enzyme Catalytic and Structural Properties. DOI:10.1021/bi9631677. PMID:9125495.
  2. Lin S et al. (2011), Mol Biosyst, 7, 1811-. Closing in on complete pathways of biotin biosynthesis. DOI:10.1039/c1mb05022b. PMID:21437340.
  3. Dey S et al. (2010), Biochemistry, 49, 6746-6760. Structural Characterization of theMycobacterium tuberculosisBiotin Biosynthesis Enzymes 7,8-Diaminopelargonic Acid Synthase and Dethiobiotin Synthetase,. DOI:10.1021/bi902097j. PMID:20565114.
  4. Sandalova T et al. (1999), Acta Crystallogr D Biol Crystallogr, 55, 610-624. Structure of dethiobiotin synthetase at 0.97 A resolution. PMID:10089457.
  5. Käck H et al. (1998), Proc Natl Acad Sci U S A, 95, 5495-5500. Snapshot of a phosphorylated substrate intermediate by kinetic crystallography. DOI:10.1073/pnas.95.10.5495. PMID:9576910.
  6. Käck H et al. (1998), Protein Sci, 7, 2560-2566. Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis. DOI:10.1002/pro.5560071209. PMID:9865950.
  7. Schneider G et al. (1997), Methods Enzymol, 376-385. [40] Structure of ATP-dependent carboxylase, dethiobiotin synthase. DOI:10.1016/s0076-6879(97)79042-6.
  8. Huang W et al. (1995), Biochemistry, 34, 10985-10995. Mechanism of an ATP-dependent carboxylase, dethiobiotin synthetase, based on crystallographic studies of complexes with substrates and a reaction intermediate. PMID:7669756.
  9. Alexeev D et al. (1995), Structure, 3, 1207-1215. Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study. PMID:8591031.
  10. Alexeev D et al. (1994), Structure, 2, 1061-1072. Mechanistic implications and family relationships from the structure of dethiobiotin synthetase. PMID:7881906.
  11. Baxter RL et al. (1994), J Chem Soc Chem Commun, 559-560. Mechanism of dethiobiotin synthetase—characterisation of the 8-aminocarbamate of (7R,8S)-7,8 diaminononanoate as an enzyme-bound intermediate. DOI:10.1039/c39940000559.
  12. Baxter RL et al. (1994), J Chem Soc Chem Commun, 0, 759-760. The mechanism of Escherichia coli dethiobiotin synthetase—the closure of the ureido ring of dethiobiotin involves formation of a carbamic-phosphate mixed anhydride. DOI:10.1039/c39940000759.
  13. Huang W et al. (1994), Structure, 2, 407-414. Crystal structure of an ATP-dependent carboxylase, dethiobiotin synthetase, at 1.65 A resolution. PMID:8081756.

Step 1. Water deprotonates the 7,8-diaminononanoate substrate, activating it for a nucleophilic attack upon the carbon dioxide in an addition reaction. Lys37 stabilises the formation of the negatively charged intermediate.

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Catalytic Residues Roles

Residue Roles
Lys37A electrostatic stabiliser, hydrogen bond donor
Ser41A increase basicity
Glu115A metal ligand
Asp54A metal ligand
Glu12A metal ligand
Thr16A metal ligand

Chemical Components

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

Step 2. The carboxylated intermediate acts as a nucleophile and attacks the gamma phosphate of ATP in a substitution reaction. Lys37, Lys15 and two Mg(II) ions stabilise the intermediates.

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Catalytic Residues Roles

Residue Roles
Lys37A hydrogen bond donor, electrostatic stabiliser
Ser41A (main-N) hydrogen bond donor
Lys15A electrostatic stabiliser, hydrogen bond donor
Thr11A electrostatic stabiliser
Ser41A electrostatic stabiliser
Glu115A metal ligand
Asp54A metal ligand
Glu12A metal ligand
Thr16A metal ligand

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation

Step 3. The newly attached phosphate group deprotonates the terminal primary amine, which in turn acts as a nucleophile to attack the phosphorylated carbonyl carbon in an internal addition reaction. Lys37, Lys15 and the main chain amide of Ser41 all stabilise the intermediates.

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Catalytic Residues Roles

Residue Roles
Lys37A hydrogen bond donor
Ser41A (main-N) hydrogen bond donor, electrostatic stabiliser
Lys15A electrostatic stabiliser, hydrogen bond donor
Thr11A electrostatic stabiliser
Ser41A electrostatic stabiliser
Glu115A metal ligand
Asp54A metal ligand
Thr16A metal ligand

Chemical Components

proton transfer, ingold: intramolecular nucleophilic addition, cyclisation, intermediate formation

Step 4. The oxyanion formed re-forms the carbonyl group, cleaving the P-O bond in a conjugate base elimination reaction. The leaving phosphate group deprotonates the newly formed secondary amine. Lys15, Lys37 and the main chain amide of Ser41 all stabilise the intermediates.

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Catalytic Residues Roles

Residue Roles
Lys37A hydrogen bond donor, electrostatic stabiliser
Lys15A hydrogen bond donor, electrostatic stabiliser
Ser41A (main-N) electrostatic stabiliser, hydrogen bond donor
Thr11A electrostatic stabiliser
Ser41A electrostatic stabiliser
Glu115A metal ligand
Asp54A metal ligand
Thr16A metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, overall product formed, dephosphorylation, intermediate terminated, intermediate collapse
Download: Image, Marvin File

Step 1. Water deprotonates the 7,8-diaminononanoate substrate, activating it for a nucleophilic attack upon the carbon dioxide in an addition reaction. Lys37 stabilises the formation of the negatively charged intermediate.

Step 2. The carboxylated intermediate acts as a nucleophile and attacks the gamma phosphate of ATP in a substitution reaction. Lys37, Lys15 and two Mg(II) ions stabilise the intermediates.

Step 3. The newly attached phosphate group deprotonates the terminal primary amine, which in turn acts as a nucleophile to attack the phosphorylated carbonyl carbon in an internal addition reaction. Lys37, Lys15 and the main chain amide of Ser41 all stabilise the intermediates.

Step 4. The oxyanion formed re-forms the carbonyl group, cleaving the P-O bond in a conjugate base elimination reaction. The leaving phosphate group deprotonates the newly formed secondary amine. Lys15, Lys37 and the main chain amide of Ser41 all stabilise the intermediates.

Products.

Contributors

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