Riboflavin Synthase

I would like to convince you that Riboflavin Synthase deserves the title of Protein of the Year due to it's beautiful structure, complex catalytic mechanism, and it's impact on fighting gram negative bacteria. 

Riboflavin Synthase 1I8D

  

Riboflavin Synthase exist in a homotrimer form.  It's structure consist of two beta barrel domains with a C-terminus alpha helix.  Each monomer then contains two active sites. However, only one riboflavin molecule can be formed at a time for each monomer as the other is forced to face outwards towards the solvent.  (1)

Riboflavin Synthase monomer 1KZL

 

 

Riboflavin Synthase monomer with Substrate 1KZL

  One of the surprising facts about this enzyme is that it is not normally found in humans.  As we are able to take in riboflavin from the environment, where as bacteria are unable to.  This has lead scientist to believe then that riboflavin synthase could be a potential antibacterial drug.  By inhibiting this enzyme, gram negative bacteria would be unable to produce riboflavin and subsequently die.  With this in mind, researchers have developed a number of inhibitors with the most effective being 9-D-ribityl-1,3,7-trihydropurine-2,6,8-purinetrione (shown below). (2)

This inhibitor works via competitive inhibition with the substrate for riboflavin synthase 6,7-dimethyl-8-ribityllumazine.  It is also important to note here that riboflavin synthase often acts as a bifunctional enzyme with lumazine synthase.  Thus, when these inhibitors were being made the researchers were looking at the effects it has on the overall complex.  (2)

Riboflavin Synthase/Lumazine Synthase Complex 1RVV

Complex in an icosahedral symmetry 1VSW

The reaction this enzyme catalyzes is:

(2) 6,7-dimethyl-8-ribityllumazine → riboflavin + 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.
The mechanism for this has been solved by researchers.  In this mechanism, a nucleophile is added to one molecule of 6,7-dimethyl-8-ribityllumazine, which then goes under nucleophilic attack.  Two elimination reactions then occur and finally the intermediate aromatizes through another elimination reaction to yield the products. (3)

N-Terminal Bonding of Riboflavin Synthase with Substrate (4)

 

C-Terminal Bonding of Riboflavin Synthase and Substrate (4)

As you can see this enzyme has a extremely complex mechanism.  However, it is necessary to the life of gram negative bacteria.  I hope you now can see the beauty of this protein, and its huge significance towards developing antibacterial drugs. 

References:
(1) Liao DI, Wawrzak Z, Calabrese JC, Viitanen PV, Jordan DB (May 2001). "Crystal structure of riboflavin synthase". Structure 9 (5): 399–408
(2) Cushman M, Yang D, Kis K, Bacher A (December 2001). "Design, synthesis, and evaluation of 9-D-ribityl-1,3,7-trihydro-2,6,8-purinetrione, a potent inhibitor of riboflavin synthase and lumazine synthase". J. Org. Chem. 66 (25): 8320–7.
(3) Fischer M, Schott AK, Kemter K, Feicht R, Richter G, Illarionov B, Eisenreich W, Gerhardt S, Cushman M, Steinbacher S, Huber R, Bacher A (December 2003). "Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments". BMC Biochem. 4: 18. 
(4)  http://en.wikipedia.org/wiki/Riboflavin_synthase                                                                                                                                               

Journal Article Summaries on Riboflavin Synthase

Liao DI, Wawrzak Z, Calabrese JC, Viitanen PV, Jordan DB (May 2001). "Crystal structure of riboflavin synthase". Structure 9 (5): 399–408

The first article I chose dealt with the overall structure of riboflavin synthase.  The protein itself exists in a homotrimer form, and requires no additional cofactors to carry out its reaction.  The enzyme is not typically found in humans, but it is very common in many bacteria.  Each monomer contains two beta barrels and a C-terminal alpha helix.  During the reaction, only one active site is available at a time.  If you’re curious to see how awesome this structure really looks just check out my previous blog post. 

Fischer M, Schott AK, Kemter K, Feicht R, Richter G, Illarionov B, Eisenreich W, Gerhardt S, Cushman M, Steinbacher S, Huber R, Bacher A (December 2003). "Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments". BMC Biochem. 4: 18.

The second article I chose explained the mechanism of the reaction in good detail.  The overall reaction done by riboflavin synthase is:  (2) 6,7-dimethyl-8-ribityllumazine → riboflavin + 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.  In this mechanism, a nucleophile is added to one molecule of 6,7-dimethyl-8-ribityllumazine, which then goes under nucleophilic attack.  Two elimination reactions then occur and finally the intermediate aromatizes through another elimination reaction to yield the products.  Overall, it is a quite complicated mechanism to understand.  It is interesting to note here that this reaction can occur without the enzyme in boiling solution.  However, bacteria need this enzyme to survive as they are unable to uptake riboflavin externally.

Cushman M, Yang D, Kis K, Bacher A (December 2001). "Design, synthesis, and evaluation of 9-D-ribityl-1,3,7-trihydro-2,6,8-purinetrione, a potent inhibitor of riboflavin synthase and lumazine synthase". J. Org. Chem. 66 (25): 8320–7.

The third article I chose discusses an inhibitor of riboflavin synthase.  As mentioned before, bacteria are unable to take up riboflavin from their environment.  With this in mind, scientist have realized that the ability to inhibit riboflavin synthase in bacteria would result in their destruction.  You can imagine how useful this would be to kill off harmful gram-negative bacteria.   The researchers in this article were able to design and synthesize a number of inhibitors with the most effective being: 9-D-Ribityl-1,3,7-trihydro-2,6,8-purinetrione.  It is also important to note here that riboflavin synthase and lumazine synthase are often found in complex with one another as lumazine synthase recycles the pyrimidinedione product from riboflavin synthase.  Many of these inhibitors found act on one or both of these enzymes.  

Riboflavin Synthase (Lumazine synthase) Pictures

Line Representation of Riboflavin Synthase

Surface Representation of Riboflavin Synthase

Icosahedral Riboflavin Synthase from Bacillus Anthracis

Sphere Representation of Riboflavin Synthase

Cartoon Representation of Riboflavin Synthase