X-ray crystallographic analysis of Lumazine Synthase from Brucella abortus bound to a substrate analogue inhibitor at 2.90 Å . (Poster)

SEBASTIÁN KLINKE; VANESA ZYLBERMAN; DANIEL R. VEGA; BEATRIZ G. GUIMARÃES; BRADFORD C. BRADEN; FERNANDO A. GOLDBAUM

Erice

Encuentro; Meeting "Evolving Methods in Macromolecular Crystallography, the 37th crystallographic meeting at Erice"; 2005

Ettore Majorana Foundation and Centre for Scientific Culture

Lumazine synthase (LS) is an enzyme which catalyses the formation of 6,7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboflavin. It is found in plants, fungi and microorganisms, showing several degrees of quaternary organization. We study this protein from Brucella abortus, the infectious organism that causes brucellosis. This protein folds as a very stable dimer of pentamers, giving rise of an overall decameric quaternary structure. It has been shown that this enzyme can bind several substrate and product analogues and also riboflavin. Our goal is to describe its active site by means of protein X-ray crystallography; for this reason we cocrystallized lumazine synthase in the presence of the substrate analogue inhibitor 5-nitro-6- (D-ribitylamino)-2,4(1H,3H)-pyrimidinedione, whose only difference with the natural substrate is a 5-nitro instead of a 5-amino group. We obtained diamond-shaped trigonal crystals which diffracted to a maximum resolution of 2.90 Å at the D03B Protein Crystallography Beamline at the Laboratório Nacional de Luz Síncrotron in Campinas, Brazil. Processing of the frames were done with the CCP4 suite. Preliminary phasing was achieved by the molecular replacement technique using previously solved free B. abortus lumazine synthase (PDB code 1DI0) as search model. We found one pentamer in the asymmetric unit with very strong electronic density near the five topologically equivalent active sites, corresponding to the bound substrate analogue inhibitor. The biological decameric arrangement can be easily built by means of the symmetry operators of the trigonal space group P3121. We obtained a final model with R=0.217 and Rfree=0.254 with goodgeometry. The substrate analogue inhibitor is recognized by the lumazine synthase by means of several welldefined hydrophilic contacts and a strong hydrophobic stacking that involve residues from two neighboring chains. We also found a significant difference in the structure of the pentamer-pentamer interface between free LS and its complexed form. Crystallographic studies confirmed that this difference is not related to ligand binding but is the consequence of different pH conditions in the crystallization. In order to have a more complete description of the LS active site, we are now trying to crystallize and solve the three-dimensional structure of LS bound to riboflavin to compare the binding mode with those of the substrate analogue inhibitor 6presented here. A proper description of the active site of this enzyme can help in the future to design useful specific inhibitor drugs for the treatment of brucellosis.