Riboflavin synthase

 

Riboflavin (vitamin B2) serves as a precursor of flavocoenzymes, which have essential roles as redox cofactors in all organisms. The final step in the biosynthesis of the vitamin is catalysed by the enzyme riboflavin synthase. This unusual reaction involves the dismutation of 6,7-dimethyl-8-ribityllumazine. A 4-carbon unit is transferred between two of the identical substrates to form riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione involving the cleavage of two C-N bonds and the formation of two C-C bonds by the transfer of a 4-carbon moiety. The three active sites of the trimer lie between pairs of monomers, although only one active site can be formed and catalytically competent at any one time. The homotrimer is non-existent in humans and is an attractive target for antimicrobial agents.

 

Reference Protein and Structure

Sequence
Q9Y7P0 UniProt (2.5.1.9) IPR001783 (Sequence Homologues) (PDB Homologues)
Biological species
Schizosaccharomyces pombe 972h- (Fission yeast) Uniprot
PDB
1kzl - Riboflavin Synthase from S.pombe bound to Carboxyethyllumazine (2.1 Å) PDBe PDBsum 1kzl
Catalytic CATH Domains
2.40.30.20 CATHdb (see all for 1kzl)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:2.5.1.9)

6,7-dimethyl-8-(1-D-ribityl)lumazine(1-)
CHEBI:58201ChEBI
+
hydron
CHEBI:15378ChEBI
→
5-amino-6-(D-ribitylamino)uracil
CHEBI:15934ChEBI
+
riboflavin(1-)
CHEBI:57986ChEBI
Alternative enzyme names: Heavy riboflavin synthase, Light riboflavin synthase, Riboflavin synthetase, Riboflavine synthase, Riboflavine synthetase,

Enzyme Mechanism

Introduction

This mechanism is not yet fully characterised, and the roles of the various amino acid residues are still very putative.

In this proposal, His102 deprotonates the 7 position methyl group of the first substrate molecule in an initial activation step. Cys48 initiates a nucleophilic attack on the second substrate in an addition reaction with the concomitant deprotonation of solvent water. (The nucleophile could be a hydroxide formed from water.) The oxyanion of the first activated substrate collapses, initiating a nucleophilic attack on the second activate substrate with concomitant deprotonation of His102. His102 deprotonates the intermediate, initiating a double bond rearrangement, re-forming the oxyanion. The intermediate tautomerises, in two steps. Water deprotonates the amine with concomitant double bond rearrangement which initiates a nucleophilic attack on the carbon to which the Cys48 is covalently attached in a substitution reaction. The oxyanion collapses, initiating double bond rearrangement that results in the cleavage of the C-N bond in an intramolecular elimination with concomitant deprotonation of His102. Cys48 deprotonates the ring CH2 group, initiating an elimination reaction, which liberates the reaction products with concomitant deprotonation of Cys48. The role of individual amino acids is still putative.

Catalytic Residues Roles

UniProt PDB* (1kzl)
Phe2 Phe2A The Phe2 side chain stabilises the Cys48 thiol so that nucleophilic attack can occur. increase nucleophilicity, enhance reactivity, electrostatic stabiliser, increase acidity
Ser41 Ser41A Aids E2 elimination by Cys48, could potentially act as a general acid. Ser41 also stabilises the Cys48 thiolate ion forming a hydrogen bond to the cysteine residue. hydrogen bond donor, electrostatic stabiliser, increase acidity, increase basicity, increase nucleophilicity
Cys48 Cys48A Cys48 thiol group nucleophilically attacks the 7-position of one of the lumazine substrate molecules, and is stabilised by hydrogen bonding to Ser 41 and Phe 2. Also acts as a general acid/base in the final steps of the mechanism in the E2 elimination that forms the products. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge, activator, increase electrophilicity
Thr50 Thr50A Thought to activate the second tautomerisation. increase basicity, hydrogen bond donor, electrostatic stabiliser, increase acidity
His102 His102A Acts in conjunction with Thr148 as a dyad in general base/acid function. Initiates formation of oxyanion via deprotonation of DMRL. Initiates tautomerisation. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Thr148 Thr148A Aids His102 in its function as a general acid/base. increase basicity, hydrogen bond acceptor, electrostatic stabiliser, increase acidity
Ala64 (main-N) Ala64A (main-N) Stabilises the exo-methylene anion by hydrogen bonding via its backbone amide. The identity of this residue doesn't seem to be conserved (Met in the E. coli protein, Ala in the S. pombe protein). 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

proton transfer, overall reactant used, intermediate formation, bimolecular nucleophilic addition, enzyme-substrate complex formation, intramolecular rearrangement, tautomerisation (not keto-enol), intermediate terminated, intramolecular nucleophilic substitution, enzyme-substrate complex cleavage, cyclisation, intramolecular elimination, decyclisation, bimolecular elimination, overall product formed, native state of enzyme regenerated

References

  1. Fischer M et al. (2008), Arch Biochem Biophys, 474, 252-265. Biosynthesis of vitamin B2: Structure and mechanism of riboflavin synthase. DOI:10.1016/j.abb.2008.02.008. PMID:18298940.
  2. Kim RR et al. (2010), J Am Chem Soc, 132, 2983-2990. Mechanistic Insights on Riboflavin Synthase Inspired by Selective Binding of the 6,7-Dimethyl-8-ribityllumazine Exomethylene Anion. DOI:10.1021/ja908395r. PMID:20143812.
  3. Fischer M et al. (2005), Nat Prod Rep, 22, 324-350. Biosynthesis of flavocoenzymes. DOI:10.1039/b210142b. PMID:16010344.
  4. Illarionov B et al. (2005), Biol Chem, 386, 127-136. Pre-steady-state kinetic analysis of riboflavin synthase using a pentacyclic reaction intermediate as substrate. DOI:10.1515/bc.2005.016. PMID:15843156.
  5. Zheng YJ et al. (2003), Bioorg Chem, 31, 278-287. Examination of a reaction intermediate in the active site of riboflavin synthase. DOI:10.1016/s0045-2068(03)00029-4. PMID:12877878.
  6. Fischer M et al. (2003), BMC Biochem, 4, 18-. Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments. DOI:10.1186/1471-2091-4-18. PMID:14690539.
  7. Liao DI et al. (2001), Structure, 9, 399-408. Crystal Structure of Riboflavin Synthase. DOI:10.1016/s0969-2126(01)00600-1. PMID:11377200.
  8. Truffault V et al. (2001), J Mol Biol, 309, 949-960. The solution structure of the N-terminal domain of riboflavin synthase. DOI:10.1006/jmbi.2001.4683. PMID:11399071.
  9. Illarionov B et al. (2001), J Biol Chem, 276, 11524-11530. Riboflavin Synthase of Escherichia coli. EFFECT OF SINGLE AMINO ACID SUBSTITUTIONS ON REACTION RATE AND LIGAND BINDING PROPERTIES. DOI:10.1074/jbc.m008931200. PMID:11278450.

Step 1. His102 deprotonates the 7 position methyl group of the first substrate molecule in an initial activation step.

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Catalytic Residues Roles

Residue Roles
Thr148A hydrogen bond acceptor, increase basicity
Ser41A hydrogen bond donor, electrostatic stabiliser
His102A hydrogen bond donor, hydrogen bond acceptor
Cys48A hydrogen bond acceptor
Thr50A hydrogen bond donor
Ala64A (main-N) hydrogen bond donor
Phe2A electrostatic stabiliser
His102A proton acceptor

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 2. Cys48 initiates a nucleophilic attack on the second substrate in an addition reaction with the concomitant deprotonation of solvent water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr148A hydrogen bond acceptor
Ser41A increase nucleophilicity, hydrogen bond donor
His102A hydrogen bond donor
Cys48A hydrogen bond acceptor
Thr50A hydrogen bond donor
Phe2A increase nucleophilicity
Ala64A (main-N) electrostatic stabiliser
Cys48A nucleophile

Chemical Components

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

Step 3. The oxyanion of the first activated substrate collapses, initiating a nucleophilic attack on the second activate substrate with concomitant deprotonation of His102.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr148A increase acidity, hydrogen bond acceptor
His102A hydrogen bond donor
Cys48A covalently attached, increase electrophilicity
Thr50A hydrogen bond donor
Ala64A (main-N) electrostatic stabiliser
His102A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, intermediate formation

Step 4. His102 deprotonates the intermediate, initiating a double bond rearrangement, re-forming the oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr148A increase basicity
His102A hydrogen bond acceptor, hydrogen bond donor
Cys48A covalently attached, activator
Thr50A hydrogen bond donor
Ala64A (main-N) electrostatic stabiliser
His102A proton acceptor

Chemical Components

proton transfer, intramolecular rearrangement, intermediate formation

Step 5. The intermediate tautomerises.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr148A hydrogen bond acceptor
His102A hydrogen bond donor
Cys48A covalently attached, activator
Thr50A increase basicity, hydrogen bond donor

Chemical Components

tautomerisation (not keto-enol), intermediate terminated

Step 6. Water deprotonates the amine with concomitant double bond rearrangement which initiates a nucleophilic attack on the carbon to which the Cys48 is covalently attached in a substitution reaction.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr148A hydrogen bond acceptor
Ser41A hydrogen bond donor, electrostatic stabiliser
His102A hydrogen bond donor
Cys48A covalently attached, hydrogen bond acceptor
Thr50A increase acidity, hydrogen bond donor
Ala64A (main-N) electrostatic stabiliser
Cys48A nucleofuge

Chemical Components

proton transfer, ingold: intramolecular nucleophilic substitution, enzyme-substrate complex cleavage, intermediate formation, cyclisation

Step 7. The oxyanion collapses, initiating double bond rearrangement that results in the cleavage of the C-N bond in an intramolecular elimination with concomitant deprotonation of His102.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr148A increase basicity, hydrogen bond acceptor
Ser41A hydrogen bond donor
His102A hydrogen bond donor
Cys48A hydrogen bond acceptor
Thr50A hydrogen bond donor
Ala64A (main-N) electrostatic stabiliser
Phe2A electrostatic stabiliser
His102A proton donor

Chemical Components

proton transfer, intramolecular rearrangement, ingold: intramolecular elimination, decyclisation, intermediate formation

Step 8. Cys48 deprotonates the ring CH2 group, initiating an elimination reaction, which liberates the reaction products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr148A hydrogen bond acceptor
Ser41A hydrogen bond donor, increase basicity
His102A hydrogen bond donor
Cys48A hydrogen bond acceptor, hydrogen bond donor
Thr50A hydrogen bond donor
Phe2A enhance reactivity
Ala64A (main-N) electrostatic stabiliser
Cys48A proton acceptor

Chemical Components

ingold: bimolecular elimination, overall product formed, proton transfer

Step 9. This step probably occurs in a concerted manner with step 8 (shown separately for clarity). The riboflavin product deprotonates Cys48 to regenerate the active site and produce the final product.

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Catalytic Residues Roles

Residue Roles
Phe2A increase acidity
Ser41A increase acidity
Thr50A electrostatic stabiliser
Ala64A (main-N) electrostatic stabiliser
Thr148A electrostatic stabiliser
Cys48A proton donor

Chemical Components

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

Step 1. His102 deprotonates the 7 position methyl group of the first substrate molecule in an initial activation step.

Step 2. Cys48 initiates a nucleophilic attack on the second substrate in an addition reaction with the concomitant deprotonation of solvent water.

Step 3. The oxyanion of the first activated substrate collapses, initiating a nucleophilic attack on the second activate substrate with concomitant deprotonation of His102.

Step 4. His102 deprotonates the intermediate, initiating a double bond rearrangement, re-forming the oxyanion.

Step 5. The intermediate tautomerises.

Step 6. Water deprotonates the amine with concomitant double bond rearrangement which initiates a nucleophilic attack on the carbon to which the Cys48 is covalently attached in a substitution reaction.

Step 7. The oxyanion collapses, initiating double bond rearrangement that results in the cleavage of the C-N bond in an intramolecular elimination with concomitant deprotonation of His102.

Step 8. Cys48 deprotonates the ring CH2 group, initiating an elimination reaction, which liberates the reaction products.

Step 9. This step probably occurs in a concerted manner with step 8 (shown separately for clarity). The riboflavin product deprotonates Cys48 to regenerate the active site and produce the final product.

Products.

Contributors

Gemma L. Holliday, Anna Waters, Craig Porter, Emma Penn