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Markus Fischer - One of the best experts on this subject based on the ideXlab platform.
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potential anti infective targets in pathogenic yeasts structure and properties of 3 4 dihydroxy 2 Butanone 4 phosphate synthase of candida albicans
Journal of Molecular Biology, 2004Co-Authors: Stefanie Echt, Adelbert Bacher, Stefan Steinbacher, Robert Huber, Stefanie Bauer, Markus FischerAbstract: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 A, 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.
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metal sites in 3 4 dihydroxy 2 Butanone 4 phosphate synthase from methanococcus jannaschii in complex with the substrate ribulose 5 phosphate
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Stefan Steinbacher, Adelbert Bacher, Susanne Schiffmann, Markus FischerAbstract: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.
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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
Journal of Biological Chemistry, 2003Co-Authors: Stefan Steinbacher, Gerald Richter, Adelbert Bacher, Susanne Schiffmann, Robert Huber, Markus FischerAbstract: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.
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Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-Butanone-4-phosphate synthase of Methanococcus jannaschii.
The Journal of biological chemistry, 2002Co-Authors: Markus Fischer, Gerald Richter, Stefan Steinbacher, Susanne Schiffmann, Robert Huber, Werner Römisch, Mark Kelly, Hartmut Oschkinat, Wolfgang Eisenreich, Adelbert BacherAbstract:Abstract 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−1min−1 at 37 °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 13C NMR spectroscopy using [U-13C5]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.
Adelbert Bacher - One of the best experts on this subject based on the ideXlab platform.
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potential anti infective targets in pathogenic yeasts structure and properties of 3 4 dihydroxy 2 Butanone 4 phosphate synthase of candida albicans
Journal of Molecular Biology, 2004Co-Authors: Stefanie Echt, Adelbert Bacher, Stefan Steinbacher, Robert Huber, Stefanie Bauer, Markus FischerAbstract: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 A, 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.
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metal sites in 3 4 dihydroxy 2 Butanone 4 phosphate synthase from methanococcus jannaschii in complex with the substrate ribulose 5 phosphate
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Stefan Steinbacher, Adelbert Bacher, Susanne Schiffmann, Markus FischerAbstract: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.
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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
Journal of Biological Chemistry, 2003Co-Authors: Stefan Steinbacher, Gerald Richter, Adelbert Bacher, Susanne Schiffmann, Robert Huber, Markus FischerAbstract: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.
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Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-Butanone-4-phosphate synthase of Methanococcus jannaschii.
The Journal of biological chemistry, 2002Co-Authors: Markus Fischer, Gerald Richter, Stefan Steinbacher, Susanne Schiffmann, Robert Huber, Werner Römisch, Mark Kelly, Hartmut Oschkinat, Wolfgang Eisenreich, Adelbert BacherAbstract:Abstract 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−1min−1 at 37 °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 13C NMR spectroscopy using [U-13C5]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.
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biosynthesis of riboflavin in plants the riba gene of arabidopsis thaliana specifies a bifunctional gtp cyclohydrolase ii 3 4 dihydroxy 2 Butanone 4 phosphate synthase
Phytochemistry, 2000Co-Authors: Stefan Herz, Sabine Eberhardt, Adelbert BacherAbstract:A cDNA segment from Arabidopsis thaliana with similarity to the ribA gene of Bacillus subtilis was sequenced. A similar gene was cloned from tomato. The open reading frame of A. thaliana was fused to the malE gene of Escherichia coli and was expressed in a recombinant E. coli strain. The recombinant fusion protein was purified and shown to have GTP cyclohydrolase II activity as well as 3,4-dihydroxy-2-Butanone 4-phosphate synthase activity. The cognate gene was amplified by polymerase chain reaction from chromosomal Arabidopsis DNA and was shown to contain six introns. Intron 4 is located in the region connecting the GTP cyclohydrolase II and 3,4-dihydroxy-2-Butanone 4-phosphate synthase domain of the putative domains catalyzing the two reaction steps. By comparison with the bacterial ribA gene, the Arabidopsis gene contains an additional 5' element specifying about 120 amino acid residues. This segment contains numerous serine and threonine residues and does not show similarity with other known sequences. The N-terminal segment is not required for catalytic activity and is likely to serve as signal sequence for import into chloroplasts.
Gerald Richter - One of the best experts on this subject based on the ideXlab platform.
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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
Journal of Biological Chemistry, 2003Co-Authors: Stefan Steinbacher, Gerald Richter, Adelbert Bacher, Susanne Schiffmann, Robert Huber, Markus FischerAbstract: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.
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Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-Butanone-4-phosphate synthase of Methanococcus jannaschii.
The Journal of biological chemistry, 2002Co-Authors: Markus Fischer, Gerald Richter, Stefan Steinbacher, Susanne Schiffmann, Robert Huber, Werner Römisch, Mark Kelly, Hartmut Oschkinat, Wolfgang Eisenreich, Adelbert BacherAbstract:Abstract 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−1min−1 at 37 °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 13C NMR spectroscopy using [U-13C5]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.
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gtp cyclohydrolase ii and 3 4 dihydroxy 2 Butanone 4 phosphate synthase are rate limiting enzymes in riboflavin synthesis of an industrial bacillus subtilis strain used for riboflavin production
Journal of Industrial Microbiology & Biotechnology, 1999Co-Authors: Markus Humbelin, A. Bacher, Gerald Richter, Harald Ritz, V Griesser, Thoralf Keller, W Schurter, M Haiker, Hp Hohmann, A P G M Van LoonAbstract:One of the proteins encoded by the riboflavin operon of Bacillus subtilis, RibA, was identified as the rate limiting enzyme in an industrial riboflavin producing strain. An additional single copy of the ribA gene was introduced into the sacB locus of the riboflavin production strain and was expressed constitutively from the medium strength vegI promoter. This led to improved riboflavin titers and yields of riboflavin on glucose of up to 25%. Both enzymatic activities of RibA, the 3,4-dihydroxy-2-Butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity.
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Biosynthesis of riboflavin: 3,4-dihydroxy-2-Butanone-4-phosphate synthase.
Methods in enzymology, 1997Co-Authors: Gerald Richter, Klaus Kis, Cornelia Krieger, R. Volk, Harald Ritz, E. Götze, A. BacherAbstract:Publisher Summary The riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, is formed by condensation of 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione with 3,4-dihydroxy-2-Butanone 4-phosphate. The structure of the carbohydrate was established relatively recently. Ribulose 5-phosphate serves as substrate for the formation of 3,4-dihydroxy-2-Butanone 4-phosphate catalyzed by the enzyme 3,4-dihydroxy-2-Butanone-4-phosphate synthase. The enzyme catalyzes the release of carbon-4 of ribulose 5-phosphate as formate, which is accompanied by a complex rearrangement reaction conducive to the formation of the product 3,4-dihydroxy-2-Butanone 4-phosphate from carbon atoms 1, 2, 3, and 5 of the substrate. 3,4-Dihydroxy-2-Butanone-4-phosphate synthase requires Mg 2+ , and the enzyme reaction can be stopped by adding ethylenediaminetetraacetic acid (EDTA). For detection, the enzyme product is converted enzymatically to 6,7-dimethyl-8-ribityllumazine or riboflavin, which can be determined by fluorescence-monitored high-performance liquid chromatography (HPLC). Lumazine synthase or the lumazine synthase/riboflavin synthase complex is required for the assay and can be prepared using the method described in the chapter.
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biosynthesis of riboflavin cloning sequencing and expression of the gene coding for 3 4 dihydroxy 2 Butanone 4 phosphate synthase of escherichia coli
Journal of Bacteriology, 1992Co-Authors: Gerald Richter, Cornelia Krieger, R. Volk, H W Lahm, U Rothlisberger, A. BacherAbstract:3,4-Dihydroxy-2-Butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-Butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-Butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-Butanone 4-phosphate synthase.
A. Bacher - One of the best experts on this subject based on the ideXlab platform.
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gtp cyclohydrolase ii and 3 4 dihydroxy 2 Butanone 4 phosphate synthase are rate limiting enzymes in riboflavin synthesis of an industrial bacillus subtilis strain used for riboflavin production
Journal of Industrial Microbiology & Biotechnology, 1999Co-Authors: Markus Humbelin, A. Bacher, Gerald Richter, Harald Ritz, V Griesser, Thoralf Keller, W Schurter, M Haiker, Hp Hohmann, A P G M Van LoonAbstract:One of the proteins encoded by the riboflavin operon of Bacillus subtilis, RibA, was identified as the rate limiting enzyme in an industrial riboflavin producing strain. An additional single copy of the ribA gene was introduced into the sacB locus of the riboflavin production strain and was expressed constitutively from the medium strength vegI promoter. This led to improved riboflavin titers and yields of riboflavin on glucose of up to 25%. Both enzymatic activities of RibA, the 3,4-dihydroxy-2-Butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity.
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Biosynthesis of riboflavin: 3,4-dihydroxy-2-Butanone-4-phosphate synthase.
Methods in enzymology, 1997Co-Authors: Gerald Richter, Klaus Kis, Cornelia Krieger, R. Volk, Harald Ritz, E. Götze, A. BacherAbstract:Publisher Summary The riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, is formed by condensation of 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione with 3,4-dihydroxy-2-Butanone 4-phosphate. The structure of the carbohydrate was established relatively recently. Ribulose 5-phosphate serves as substrate for the formation of 3,4-dihydroxy-2-Butanone 4-phosphate catalyzed by the enzyme 3,4-dihydroxy-2-Butanone-4-phosphate synthase. The enzyme catalyzes the release of carbon-4 of ribulose 5-phosphate as formate, which is accompanied by a complex rearrangement reaction conducive to the formation of the product 3,4-dihydroxy-2-Butanone 4-phosphate from carbon atoms 1, 2, 3, and 5 of the substrate. 3,4-Dihydroxy-2-Butanone-4-phosphate synthase requires Mg 2+ , and the enzyme reaction can be stopped by adding ethylenediaminetetraacetic acid (EDTA). For detection, the enzyme product is converted enzymatically to 6,7-dimethyl-8-ribityllumazine or riboflavin, which can be determined by fluorescence-monitored high-performance liquid chromatography (HPLC). Lumazine synthase or the lumazine synthase/riboflavin synthase complex is required for the assay and can be prepared using the method described in the chapter.
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biosynthesis of riboflavin cloning sequencing and expression of the gene coding for 3 4 dihydroxy 2 Butanone 4 phosphate synthase of escherichia coli
Journal of Bacteriology, 1992Co-Authors: Gerald Richter, Cornelia Krieger, R. Volk, H W Lahm, U Rothlisberger, A. BacherAbstract:3,4-Dihydroxy-2-Butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-Butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-Butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-Butanone 4-phosphate synthase.
Stefan Steinbacher - One of the best experts on this subject based on the ideXlab platform.
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potential anti infective targets in pathogenic yeasts structure and properties of 3 4 dihydroxy 2 Butanone 4 phosphate synthase of candida albicans
Journal of Molecular Biology, 2004Co-Authors: Stefanie Echt, Adelbert Bacher, Stefan Steinbacher, Robert Huber, Stefanie Bauer, Markus FischerAbstract: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 A, 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.
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metal sites in 3 4 dihydroxy 2 Butanone 4 phosphate synthase from methanococcus jannaschii in complex with the substrate ribulose 5 phosphate
Acta Crystallographica Section D-biological Crystallography, 2004Co-Authors: Stefan Steinbacher, Adelbert Bacher, Susanne Schiffmann, Markus FischerAbstract: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.
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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
Journal of Biological Chemistry, 2003Co-Authors: Stefan Steinbacher, Gerald Richter, Adelbert Bacher, Susanne Schiffmann, Robert Huber, Markus FischerAbstract: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.
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Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-Butanone-4-phosphate synthase of Methanococcus jannaschii.
The Journal of biological chemistry, 2002Co-Authors: Markus Fischer, Gerald Richter, Stefan Steinbacher, Susanne Schiffmann, Robert Huber, Werner Römisch, Mark Kelly, Hartmut Oschkinat, Wolfgang Eisenreich, Adelbert BacherAbstract:Abstract 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−1min−1 at 37 °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 13C NMR spectroscopy using [U-13C5]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.