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Catalytic Site Atlas

CSA LITERATURE entry for 1i8d

E.C. nameriboflavin synthase
SpeciesEscherichia coli (Bacteria)
E.C. Number (IntEnz) 2.5.1.9
CSA Homologues of 1i8d1kzl,
CSA Entries With UniProtID P0AFU8
CSA Entries With EC Number 2.5.1.9
PDBe Entry 1i8d
PDBSum Entry 1i8d
MACiE Entry 1i8d

Literature Report

IntroductionRiboflavin 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.
MechansimRiboflavin 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.
Reaction

Catalytic Sites for 1i8d

Annotated By Reference To The Literature - Site 4 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
HisA102102macie:sideChainHis 102 carries out the second deprotonation on the methyl group at the 7-position of the substrate.
CysA4848macie:sideChainhe 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
MetA6464macie:mainChainAmideMet 64 stabilises the exo-methylene anion by hydrogen bonding via its backbone amide.
PheA22macie:sideChainThe Phe 2 side chain stabilises the Cys 48 thiol so that nucleophilic attack can occur.
SerA4141macie:sideChainSer 41 stabilises the Cys 48 thiolate ion forming a hydrogen bond to the cysteine residue.

Annotated By Reference To The Literature - Site 5 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
HisB102102macie:sideChainHis 102 carries out the second deprotonation on the methyl group at the 7-position of the substrate.
CysB4848macie:sideChainhe 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
MetB6464macie:mainChainAmideMet 64 stabilises the exo-methylene anion by hydrogen bonding via its backbone amide.
PheB22macie:sideChainThe Phe 2 side chain stabilises the Cys 48 thiol so that nucleophilic attack can occur.
SerB4141macie:sideChainSer 41 stabilises the Cys 48 thiolate ion forming a hydrogen bond to the cysteine residue.

Annotated By Reference To The Literature - Site 6 (Perform Site Search)
ResidueChainNumberUniProtKB NumberFunctional PartFunctionTargetDescription
HisC102102macie:sideChainHis 102 carries out the second deprotonation on the methyl group at the 7-position of the substrate.
CysC4848macie:sideChainhe 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
MetC6464macie:mainChainAmideMet 64 stabilises the exo-methylene anion by hydrogen bonding via its backbone amide.
PheC22macie:sideChainThe Phe 2 side chain stabilises the Cys 48 thiol so that nucleophilic attack can occur.
SerC4141macie:sideChainSer 41 stabilises the Cys 48 thiolate ion forming a hydrogen bond to the cysteine residue.

Literature References

Notes:
Liao DI
Crystal structure of riboflavin synthase.
Structure 2001 9 399-408
PubMed: 11377200
Truffault V
The solution structure of the N-terminal domain of riboflavin synthase.
J Mol Biol 2001 309 949-960
PubMed: 11399071
Illarionov B
Riboflavin synthase of Escherichia coli. Effect of single amino acid substitutions on reaction rate and ligand binding properties.
J Biol Chem 2001 276 11524-11530
PubMed: 11278450
Illarionov B
Pre-steady-state kinetic analysis of riboflavin synthase using a pentacyclic reaction intermediate as substrate.
Biol Chem 2005 386 127-136
PubMed: 15843156
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