Publications

• Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii.

  Fischer M., Roemisch W., Schiffmann S., Kelly M., Oschkinat H., Steinbacher S., Huber R., Eisenreich W., Richter G., Bacher A.

  The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the ribo ... >> More

  The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity. << Less

  J. Biol. Chem. 277:41410-41416(2002) [PubMed] [EuropePMC]

• Studies on the 4-carbon precursor in the biosynthesis of riboflavin. Purification and properties of L-3,4-dihydroxy-2-butanone-4-phosphate synthase.

  Volk R., Bacher A.

  The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A. (1985) Biochem. Biophys. Res. Commun. 127, 175-18 ... >> More

  The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A. (1985) Biochem. Biophys. Res. Commun. 127, 175-181). The enzyme catalyzing the formation of Compound X has been purified about 600-fold from the cell extract of the flavinogenic yeast Candida guilliermondii by chromatographic procedures. The purified protein appeared homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of a single polypeptide of 24 kDa. The committed substrate of the enzyme was identified as D-ribulose 5-phosphate. The enzyme yields two products which were identified as L-3,4-dihydroxy-2-butanone 4-phosphate and formate by NMR and CD spectroscopy. Mg2+ is required for activity. << Less

  J Biol Chem 265:19479-19485(1990) [PubMed] [EuropePMC]

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

  Liao D.-I., Calabrese J.C., Wawrzak Z., Viitanen P.V., Jordan D.B.

  <h4>Background</h4>3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton r ... >> More

  <h4>Background</h4>3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg(2+) for activity.<h4>Results</h4>The first three-dimensional structure of the enzyme was determined at 1.4 A resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an alpha + beta fold having a complex linkage of beta strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks.<h4>Conclusions</h4>A proposed active site was identified on the basis of amino acid 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, which may be used for recognizing the phosphate group of the substrate, the active 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 Mg(2+) cofactor within the active site. << Less

  Structure 9:11-18(2001) [PubMed] [EuropePMC]

• Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism.

  Steinbacher S., Schiffmann S., Richter G., Huber R., Bacher A., Fischer M.

  Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crys ... >> More

  Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed. << Less

  J. Biol. Chem. 278:42256-42265(2003) [PubMed] [EuropePMC]

• Potential anti-infective targets in pathogenic yeasts: structure and properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans.

  Echt S., Bauer S., Steinbacher S., Huber R., Bacher A., Fischer M.

  A synthetic gene specifying a putative 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans directed the synthesis of a 22.5 kDa peptide in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity by two chromatographic steps and was shown to ... >> More

  A synthetic gene specifying a putative 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans directed the synthesis of a 22.5 kDa peptide in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity by two chromatographic steps and was shown to catalyze the formation of L-3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 332 nmol mg(-1) min(-1). Hydrodynamic studies indicated a native molecular mass of 41 kDa in line with a homodimer structure. The protein was crystallized in its apoform. Soaking yielded crystals in complex with the substrate ribulose 5-phosphate. The structures were solved at resolutions of 1.6 and 1.7 angstroms, respectively, using 3,4-dihydroxy-2-butanone 4-phosphate synthase of E. coli for molecular replacement. Structural comparison with the orthologs of Magnaporthe grisea and Methanococcus jannaschii revealed a hitherto unknown conformation of the essential acidic active-site loop. << Less

  J. Mol. Biol. 341:1085-1096(2004) [PubMed] [EuropePMC]

• The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site.

  Kelly M.J.S., Ball L.J., Krieger C., Yu Y., Fischer M., Schiffmann S., Schmieder P., Kuehne R., Bermel W., Bacher A., Richter G., Oschkinat H.

  Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tai ... >> More

  Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure-activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics. Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis. Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution. << Less

  Proc. Natl. Acad. Sci. U.S.A. 98:13025-13030(2001) [PubMed] [EuropePMC]

• Metal sites in 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with the substrate ribulose 5-phosphate.

  Steinbacher S., Schiffmann S., Bacher A., Fischer M.

  The crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal centre has recently been determined at 1.7 A resolution. The enzyme converts ribulose 5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphat ... >> More

  The crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal centre has recently been determined at 1.7 A resolution. The enzyme converts ribulose 5-phosphate into 3,4-dihydroxy-2-butanone 4-phosphate, while its C4 atom is released as formate. The resulting four-carbon body supplies all eight C atoms for the xylene moiety of riboflavin. Three of the four hydroxyl groups of ribulose 5-phosphate were coordinated by the metal ions. Based on crystallographic refinement, the metals were assigned as zinc and calcium, which were present in the crystallization buffer. Neither metal supports the enzymatic reaction. In the present study, the correctness of this assignment is assessed using anomalous diffraction data collected at the high-energy side of the zinc absorption edge (lambda = 1.2823 A). Only the three tentative zinc ions give strong peaks in an anomalous difference Fourier map (>20sigma), whereas the four tentative calcium ions do not show anomalous signals above the noise level. These results confirm the initial assignment. In addition, the resolution was improved to 1.55 A. << Less

  Acta Crystallogr. D 60:1338-1340(2004) [PubMed] [EuropePMC]

• Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase.

  Liao D.-I., Zheng Y.-J., Viitanen P.V., Jordan D.B.

  X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO(4)(2-), E-SO(4)(2-)-Mg(2+), E-SO(4)(2)(-)-Mn(2+), E-SO(4)(2)(-)-Mn(2+)-glycerol, and E-SO(4)(2)(-)-Zn(2+) complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, ... >> More

  X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO(4)(2-), E-SO(4)(2-)-Mg(2+), E-SO(4)(2)(-)-Mn(2+), E-SO(4)(2)(-)-Mn(2+)-glycerol, and E-SO(4)(2)(-)-Zn(2+) complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 A, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg(2+) cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg(2+)-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme. << Less

  Biochemistry 41:1795-1806(2002) [PubMed] [EuropePMC]