Structural study on the enzyme riboflavin synthase from the pathogenic bacterium Brucella abortus

SEBASTIAN KLINKE; MARÍA I SERER; BEATRIZ G. GUIMARÃES; FERNANDO A. GOLDBAUM

Santa Fe

Congreso; VIII Reunión Anual de la Asociación Argentina de Cristalografía; 2012

Asociación Argentina de Cristalografía

The enzymes that catalyze the biosynthesis of riboflavin (vitamin B2) in pathogenic bacteria are promising targets for the development of chemotherapeutic agents. This arises from the facts that i) these microorganisms cannot incorporate exogenous riboflavin, and ii) animals lack the required enzymes and must acquire this vitamin exclusively through diet. The last step in the biosynthesis of riboflavin is catalyzed by riboflavin synthase (RS), which binds two molecules of 6,7-dimethyl-8-ribityllumazine and facilitates their dismutation into one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. In this work, we present a detailed structural description of RS from the pathogenic bacterium Brucella abortus. This microorganism is the causative agent of the disease brucellosis, a worldwide zoonosis that affects cattle and that is responsible for huge economic losses in South America.We were able to crystallize RS and solve its structure in four different conditions: i) as apo enzyme (without ligands), ii) bound to riboflavin, iii) bound to the product analogue roseoflavin, and iv) bound to the product analogue 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The overall structure shows a trimeric quaternary association, in which the monomers are organized in an asymmetric manner. Interestingly, each of the monomers bears two binding sites for the substrate 6,7-dimethyl-8-ribityllumazine, yet there is only one competent active site in the whole trimer, which is located at the interface between two neighboring monomers. The recognition of the bound ligands by the enzyme and the role of its key residues in the catalytic process will be described in detail. Additionally, a comparison between RS from Brucella abortus and Escherichia coli (the only trimeric RS with previously known crystal structure) will be presented. At the moment, catalytic activity as well as inhibitor displacement experiments are being performed to complement the crystallographic study. A proper description of the active site of this enzyme may lead to the rational design of powerful antibacterial compounds against Brucella. This work was supported by CONICET and ANPCyT. We acknowledge access to the X-ray facilities at the Institut Pasteur Montevideo (Uruguay), the National Synchrotron Light Source (USA) and the SOLEIL Synchrotron (France).