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

Angra dos Reis, estado de Río de Janeiro, Brasil

Congreso; 1st Latin American Protein Society Meeting (LAPSM); 2004

Protein Society

Lumazine synthase (LS) is an enzyme which catalyses the formation of 6,7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboavin. 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. Previous crystallization and solution studies demonstrated that 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 and therefore it remains stacked in the active site. We obtained diamond-like trigonal crystals which diffracted to a maximum resolution of 2.90A at the D03B Protein Crystallography Beamline at the Laboratorio Nacional de Luz Sincrotron in Campinas, Brazil. Indexing, integration and scaling of the frames were done with the CCP4 suite. Preliminary phasing was achieved by the molecular replacement technique using previously solved wild type 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 P3121 space group. After several cycles of positional and B-factor refinement together with visual building, we could obtain a final model with R=0.217 and Rfree=0.255 with good geometry. The substrate analogue inhibitor is recognized by the lumazine synthase by means of several well-defined hydrophilic contacts and a strong hydrophobic stacking that involves residues not only from the same monomer but also from the neighboring polypeptide chain. Moreover, we could see a very strong difference in the structure of the pentamer-pentamer interface between the wild type LS and its complexed form that we present in this work. In the latter, there is a marked decrease of the B-factor values of the residues involved in the interface, as well as an increase in the number of interatomic contacts in this area. These preliminary results indicate that the observed changes would be a consequence of the substrate analogue binding, whose positions in this oligomeric protein are quite near the pentamer-pentamer interface. 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 riboavine to compare the binding mode with those of the substrate analogue inhibitor presented in this work. 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.