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A. Bacher - One of the best experts on this subject based on the ideXlab platform.
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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. Studies on the mechanism of L-3,4-dihydroxy-2-butanone 4-phosphate synthase.
The Journal of biological chemistry, 1991Co-Authors: R. Volk, A. BacherAbstract:Abstract The riboflavin precursor, L-3,4-dihydroxy-2-butanone 4-phosphate, is formed from D-ribulose 5-phosphate by a single 24-kDa enzyme. Studies with various specifically 13C-labeled D-ribulose 5-phosphates as substrate showed that the carbon atoms 1-3 of the enzyme product correspond to carbon atoms 1-3 of the substrate, whereas C-4 of the product stems from C-5 of the substrate. Carbon atom 4 of the substrate is released as formate together with the hydrogen atom attached to it. The skeletal rearrangement which leads to the loss of C-4 and the direct linkage between C-3 and C-5 of the substrate is an intramolecular reaction. The hydrogen atom at C-3 of the enzyme product is introduced from solvent water. A reaction mechanism which is in agreement with all experimental data is proposed.
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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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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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.
Gerald Richter - One of the best experts on this subject based on the ideXlab platform.
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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: 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.
Soo Jin Yeom - One of the best experts on this subject based on the ideXlab platform.
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production of l ribose from l ribulose by a triple site variant of mannose 6 phosphate isomerase from geobacillus thermodenitrificans
Applied and Environmental Microbiology, 2012Co-Authors: Yuri Lim, Soo Jin YeomAbstract:A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) for l-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/liter l-ribose from 300 g/liter l-ribulose for 60 min, with a volumetric productivity of 213 g liter−1 h−1, which was 4.5-fold higher than that of the wild-type enzyme. The kcat/Km and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.
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characterization of a mannose 6 phosphate isomerase from thermus thermophilus and increased l ribose production by its r142n mutant
Applied and Environmental Microbiology, 2011Co-Authors: Soo Jin Yeom, Deok-kun OhAbstract:Optically pure carbohydrates are important precursors for pharmaceutical, food, and agrochemical products (22). Among carbohydrates, l-enantiomers have been widely used as antiviral nucleoside analogue drugs in the treatment of severe viral diseases due to their potent biological activities and lower toxicity than the corresponding d-nucleosides (3). l-Ribose, a pentose sugar, can be used as a precursor for the synthesis of antiviral drugs, such as l-nucleoside derivatives (2, 5, 14). l-Ribose can be synthesized by chemical methods from l-arabinose (1, 6, 9), l-xylose (13), d-glucose (15), d-galactose (19), d-ribose (26), or d-mannono-1,4-lactone (20). However, chemical synthesis has several disadvantages, including multiple steps, by-product formation, and chemical waste production. Recently, the enzymatic production of l-ribose has been investigated using l-arabinose (7) or l-ribulose (25). l-Ribose has been produced primarily from the cheap sugar l-arabinose because l-ribulose is an expensive sugar. An l-arabinose isomerase mutant of Escherichia coli (4) and a d-xylose isomerase mutant of Actinoplanes missouriensis (17) converted l-arabinose to l-ribose by a two-step isomerization reaction with low productivity. A recombinant E. coli strain containing l-arabinose isomerase and l-ribose isomerase (7) and purified l-arabinose isomerase and mannose-6-phosphate isomerase from Geobacillus thermodenitrificans (24) were used to produce l-ribose from l-arabinose via l-ribulose with high productivity. However, a rate-limiting step in the two enzyme systems is the conversion of l-ribulose to l-ribose using l-ribose isomerase (7, 12) or mannose-6-phosphate isomerase (23-25). Thus, biotechnological production of l-ribose has been focused on these enzymes. Mannose-6-phosphate isomerase from G. thermodenitrificans exhibits the highest activity to date for l-ribose production. Greater efficiency can be attained only through the discovery or synthesis of l-ribose-producing enzymes with higher kcat/Km. Increases in the kcat/Km ratio can be realized by genetic improvements via directed evolution and by structural modification of the determinant residues at or near the active site, based on homology models or the determined structure of the enzymes. In this study, the activities of a recombinant mannose-6-phosphate isomerase from Thermus thermophilus with different metal ions, pHs, and temperatures for l-ribulose isomerization and its substrate specificities for various aldoses and ketoses were characterized. Mutational analyses were performed with predicted active-site residues obtained from homology studies; the R142N mutant was selected as an effective l-ribose producer. The specific activity, kcat/Km, and conversion for l-ribulose using the R142N mutant were determined.
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characterization of a mannose 6 phosphate isomerase from thermus thermophilus and increased l ribose production by its r142n mutant
Applied and Environmental Microbiology, 2011Co-Authors: Soo Jin Yeom, Eunsun Seo, Bina Kim, Yeongsu KimAbstract:ABSTRACT An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for l-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of l-ribulose to l-ribose, a potential starting material for many l-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in l-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) for l-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The kcat/Km of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported kcat/Km. The R142N mutant enzyme produced 213 g/liter l-ribose from 300 g/liter l-ribulose for 2 h, with a volumetric productivity of 107 g liter−1 h−1, which was 1.5-fold higher than that of the wild-type enzyme.
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substrate specificity of a mannose 6 phosphate isomerase from bacillus subtilis and its application in the production of l ribose
Applied and Environmental Microbiology, 2009Co-Authors: Soo Jin Yeom, Namhee Kim, Chang-su ParkAbstract:The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co2+. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter−1 h−1.
