Crystal structure of lumazine synthase from Mycobacterium tuberculosis as a target for rational drug design: binding mode of a new class of purinetrione inhibitors.
The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range.
Morgunova
Ekaterina
E
Karolinska Institutet, NOVUM, Centre for Structural Biochemistry, S-14157 Huddinge, Sweden. katja.morgunova@biosci.ki.se
Meining
Winfried
W
Illarionov
Boris
B
Haase
Ilka
I
Jin
Guangyi
G
Bacher
Adelbert
A
Cushman
Mark
M
Fischer
Markus
M
Ladenstein
Rudolf
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D017433:Protein Structure, Secondary; D012256:Riboflavin / Q000096:biosynthesis; D012258:Riboflavin Synthase / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D009097:Multienzyme Complexes; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012258:Riboflavin Synthase; D012256:Riboflavin