Crystal structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase of riboflavin biosynthesis

Background: 3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commi tment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-pho sphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangem ent, and formate elimination. The enzyme is absent in humans and an attract ive target for the discovery of antimicrobials for pathogens incapable of a cquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa su bunits requires Mg2+ for activity. Results: The first three-dimensional structure of the enzyme was determined at 1.4A-angstrom resolution using the multiwavelength anomalous diffractio n (MAD) method on Escherichia coli protein crystals containing gold. The pr otein consists of an alpha + Beta fold having a complex linkage of beta str ands. Intersubunit contacts are mediated by numerous hydrophobic interactio ns and three hydrogen bond networks. Counclusions: A proposed active site was identified on the basis of amino a cid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, whi ch may be used for recognizing the phosphate group of the substrate, the ac tive site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg2+ cofactor within the active site.