Structural and biochemical studies of Riboflavin and Lumazine Synthases from Brucella abortus

SERER I.; BONOMI H.R.; KLINKE S.; GOLDBAUM F.A.

Ciudad de Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2012

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

The last two steps in the biosynthesis of riboflavin are sequentially catalyzed by Lumazine Synthase (RibH) and Riboflavin Synthase (RibE). Certain Gram-negative pathogenic bacteria like Brucella spp. lack a riboflavin uptake system and are dependent on its endogenous biosynthesis. Additionally, mammals lack the riboflavin pathway enzymes and must obtain this vitamin from their dietary. Therefore, inhibitors of riboflavin biosynthesis will display selective cytotoxicity for pathogenic microorganisms as opposed to human cells. For these reasons RibH and RibE have been identified as promising targets for the development of new antimicrobial agents for the treatment of Brucellosis. In this work we performed a screening of a library of inhibitors on RibH2 and RibE of B. abortus and we identified two putative inhibitors. By binding assays we demonstrate that these compounds are able to displace riboflavin from de RibE and RibH active sites. On the other hand, enzymatic activity assays indicate that RibE possesses non-Michaelian kinetics. Furthermore we crystallized RibE as a free enzyme and in complex with riboflavin, roseoflavin and NRP (a putative inhibitor). These newly determined structures and the biochemical characterization of RibE may be useful for the rational design of novel inhibitors with activity against Brucella.