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Catalytic Site Atlas Version 2.2.12
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CSA entry for 1i8d
Original Entry
Title:
Transferase
Compound:
Riboflavin synthase
Mutant:
No
UniProt/Swiss-Prot:
P29015-RISA_ECOLI
EC Class:
2.5.1.9
Other CSA Entries:
Overview of all sites for 1i8d
Homologues of 1i8d
Entries for UniProt/Swiss-Prot: P29015
Entries for EC: 2.5.1.9
Other Databases:
PDB entry: 1i8d
PDBsum entry: 1i8d
UniProt/Swiss-Prot: P29015
IntEnz entry: 2.5.1.9
Literature Report:
Introduction:
Riboflavin synthase catalyses the final step in the biosynthesis of riboflavin. This 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.

The homotrimer is non-existent in humans and is an attractive target for antimicrobial agents.

Mechanism:
Riboflavin synthase catalyses the dismutation of 2 molecules of 6,7-dimethyl-8-ribityllumazine to yield riboflavin and a 4-ribityl-5-amino-2,6-dihydroxypyrimidine. The reaction involves the cleavage of two CN bonds and the formation of two CC 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.

There are a number of proposed mechanisms for this reaction, however the most widely accepted hypothesis is that involving a nucleophilic Cys residue and a pentacyclic intermediate.

1. Reversible deprotonation at C7 position of lumazine occurs, prior to deprotonation at position 6 by an unknown general base, forming of an exo-methylene anion.
2. This anion is stabilised by hydrogen bonding to the amide groups of enzyme residues, such as Met 64.
3. The acidity of the 7 position of a second lumazine activates it towards nucleophilic attack by the thiol group of a well-positioned Cys 48 residue. The thiol is stabilised for deprotonation by hydrogen bonding to a neighbouring Ser 41 hydroxyl. Phe 2 stabilises the position of the Cys 48 thiol group since there is an appropriate distance between the CE1 of Phe 2 and the SG of Cys 48.
4. The exomethylene-type lumazide anion attacks the adduct of the second substrate molecule and a nucleophilic Cys 48, forming a pentacylcic intermediate.
5. A second deprotonation of the methyl group at the 7 position by His 102 occurs, alongside protonation of N1.
6. Tautomerisation occurs, with nucleophilic substitution of Cys 48 , followed by eliminations at N5* and N6* to yield the products.
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Literature reference 

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
PHEA 2 2Sidechain
ElectrostaticResidue
The Phe 2 side chain stabilises the Cys 48 thiol so that nucleophilic attack can occur.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11377200 Current protein Mutagenesis of residue
PubMed ID 15843156 Current protein Mutagenesis of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
SERA 41 41Sidechain
ElectrostaticResidue
Ser 41 stabilises the Cys 48 thiolate ion forming a hydrogen bond to the cysteine residue.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11278450 Current protein Mutagenesis of residue
PubMed ID 11377200 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
CYSA 48 48Sidechain
NucleophileSubstrate
ElectrostaticResidue
he Cys 48 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
Evidence from paper Evidence concerns Evidence type
PubMed ID 11377200 Current protein Conservation of residue
PubMed ID 11399071 Current protein Conservation of residue
PubMed ID 11399071 Current protein Residue is positioned appropriately (ligand position known)
PubMed ID 11377200 Current protein Residue is positioned appropriately (ligand position known)

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
META 64 64Backbone amide
ElectrostaticSubstrate
Met 64 stabilises the exo-methylene anion by hydrogen bonding via its backbone amide.
Evidence from paper Evidence concerns Evidence type
PubMed ID 11399071 Current protein Structural similarity to homologue of known mechanism
PubMed ID 11399071 Current protein Conservation of residue

ResidueChainNumberUniProt numberFunctional part FunctionTargetDescription
HISA 102 102Sidechain
Acid/baseSubstrate
His 102 carries out the second deprotonation on the methyl group at the 7-position of the substrate.
Evidence from paper Evidence concerns Evidence type
PubMed ID 15843156 Current protein Mutagenesis of residue
PubMed ID 11377200 Current protein Conservation of residue
PubMed ID 11278450 Current protein Mutagenesis of residue
PubMed ID 11399071 Current protein Conservation of residue
Notes:

References:
1
Crystal structure of riboflavin synthase.
D. I. Liao and Z. Wawrzak and J. C. Calabrese and P. V. Viitanen and D. B. Jordan
Structure 9, (5) 399-408, (2001).
11377200
2
The solution structure of the N-terminal domain of riboflavin synthase.
V. Truffault and M. Coles and T. Diercks and K. Abelmann and S. Eberhardt and H. Lüttgen and A. Bacher and H. Kessler
J Mol Biol 309, (4) 949-60, (2001).
11399071
3
Riboflavin synthase of Escherichia coli. Effect of single amino acid substitutions on reaction rate and ligand binding properties.
B. Illarionov and K. Kemter and S. Eberhardt and G. Richter and M. Cushman and A. Bacher
J Biol Chem 276, (15) 11524-30, (2001).
11278450
4
Pre-steady-state kinetic analysis of riboflavin synthase using a pentacyclic reaction intermediate as substrate.
B. Illarionov and I. Haase and M. Fischer and A. Bacher and N. Schramek
Biol Chem 386, (2) 127-36, (2005).
15843156
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