The enzymes involved in riboflavin biosynthesis are considered to be potential anti-bacterial drug targets because these proteins are essential in bacterial pathogens but are absent in humans. 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) is one of the key enzymes in the biosynthesis of riboflavin. DHBPS catalyzes the conversion of ribulose-5-phosphate (Ru5P) to 3,4-Dihydroxy-2-butanone-4-phosphate (DHBP) and formate. The purified SpDHBPS enzyme, in the presence of Mg2+ ion, catalyzed the conversion of Ru5P to DHBP at a rate of 109 nmol min(-1) mg(-1) with an apparent K-m value of 181 mu M at 37 degrees C. Surprisingly, our experiments first revealed that DHBPS showed activity in the presence of the trivalent metal ion, Fe3+. Furthermore, we determined the crystal structure of DHBPS from Gram-positive bacteria, Streptococcus pneumoniae, with 2.0 angstrom resolution. The overall architecture of SpDHBPS was similar to its homologs, which comprise one beta-sheet (five-stranded) and eight alpha-helices, adopting a three-layered alpha-beta-alpha sandwich fold. Similar to the homologs, gel-filtration experiments verified that the enzyme was arranged as a dimer. Although the overall fold of DHBPS was similar, the significant structural differences between the species at the active site region may be utilized to develop antibacterial agents that are species-specific. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.