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Fred C Hartman - One of the best experts on this subject based on the ideXlab platform.
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facilitation of the terminal proton transfer reaction of Ribulose 1 5 bisphosphate carboxylase oxygenase by active site lys166
Biochemistry, 1996Co-Authors: Mark R Harpel, Fred C HartmanAbstract:The terminal step in the carboxylation pathway catalyzed by Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is stereospecific protonation of the C-2 aci-acid of 3-phosphoglycerate (PGA). ...
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beta elimination of phosphate from reaction intermediates by site directed mutants of Ribulose bisphosphate carboxylase oxygenase
Journal of Biological Chemistry, 1994Co-Authors: Frank W Larimer, Mark R Harpel, Fred C HartmanAbstract:Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodospirillum rubrum Ribulose-bisphosphate carboxylase/oxygenase are positioned to serve as hydrogen-bond donors for the C1 phosphate of Ribulose bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Branden, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functional contributions of the residues implicated in Ribulose bisphosphate binding and intermediate stabilization, we have replaced them individually with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on enolization activity), increase of Km (Ribulose bisphosphate), and decrease of carboxylation: oxygenation specificity. During catalytic turnover of Ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, reflecting beta-elimination of phosphate from the enediol(ate) intermediate. This side product is not observed with wild-type enzyme, nor has it been reported with mutant enzymes characterized previously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the acicarbanion of 3-phosphoglycerate (the terminal intermediate). These data provide direct evidence that phosphate ligands stabilize conformations of intermediates that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.
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perturbation of reaction intermediate partitioning by a site directed mutant of Ribulose bisphosphate carboxylase oxygenase
Journal of Biological Chemistry, 1993Co-Authors: Eva H Lee, Mark R Harpel, Yuhru Chen, Fred C HartmanAbstract:Abstract To explore the roles of active-site Glu48 of Ribulose-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, the E48Q mutant has been characterized with respect to kinetics and product distribution. Although the kcat for carboxylase activity is only 0.6% of the wild-type value, the mutant retains full activity in catalyzing the conversion of the carboxylated reaction intermediate to 3-phosphoglycerate and retains 10% of the normal activity in catalyzing the enolization of Ribulose bisphosphate. Thus, the mutant is preferentially impaired in the carboxylation step. Partitioning of the enediol(ate) intermediate during turnover of Ribulose bisphosphate is perturbed dramatically in the case of the mutant protein. Whereas the wild-type enzyme displays a CO2/O2 specificity factor of 11, the corresponding parameter of the mutant is only 0.3, thereby signifying a shift of the relative reactivity of the enediol(ate) in favor of O2. The mutant protein is also unable to protect the enediol(ate) against misprotonation with consequential conversion of Ribulose bisphosphate to xylulose bisphosphate. This side reaction, undetected with wild-type R. rubrum enzyme, proceeds as rapidly as carboxylation of D-Ribulose 1,5-bisphosphate by the E48Q mutant. Formation of xylulose bisphosphate by the mutant does not appear to account for the decline in carboxylase activity that occurs during the course of an assay. These studies demonstrate the multiple functionalities of Glu48 in the facilitation of catalysis and in directing intermediate partitioning in the preferred direction.
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, Changsu 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.
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Novel substrates of a ribose-5-phosphate isomerase from Clostridium thermocellum.
Journal of Biotechnology, 2008Co-Authors: Ran-young Yoon, Soo Jin Yeom, Deok-kun OhAbstract:A substrate specificity study of the recombinant D-ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum was performed. Among all aldopentoses and aldohexoses, the RpiB enzyme displayed activity with L-talose, D-ribose, D-allose, L-allose, L-ribose, and D-talose in decreasing order. The products released were L-tagatose, D-Ribulose, D-psicose, L-psicose, L-Ribulose, and D-tagatose, respectively. The enzyme showed specificity for aldose substrates possessing hydroxyl groups oriented in the same direction at the C2, C3, and C4 positions. Molecular modeling of the enzyme suggests that the novel substrate specificity may be explained by substrate interactions with residues Tyr42, His98, and His9, which interact with the hydroxyl groups of C2, C3, and C4, respectively, oriented in the same direction. L-Talose and D-Ribulose exhibited the highest activity among the aldoses and ketoses, respectively. Ribose 5-phosphate isomerase catalyzed the conversion of L-talose to L-tagatose with an 89% conversion yield after approximately 90 min, while D-Ribulose was converted to D-ribose with a 38% conversion yield.
Markus Fischer - One of the best experts on this subject based on the ideXlab platform.
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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, Adelbert Bacher, Susanne Schiffmann, Gerald Richter, 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.
F R Tabita - One of the best experts on this subject based on the ideXlab platform.
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high substrate specificity factor Ribulose bisphosphate carboxylase oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial rubisco containing algal residue modifications
Archives of Biochemistry and Biophysics, 1994Co-Authors: Betsy A Read, F R TabitaAbstract:Marine algae play an important role in removing carbon dioxide from the atmosphere. In this investigation, we have determined the substrate specificity factor of Ribulose 1,5-bisphosphate carboxylase/oxygenase from several marine chromophytic and rhodophytic algae. The enzymes were purified to homogeneity and all possessed significantly higher substrate specificity factors than the enzymes from terrestrial plants, green algae, or bacteria. There are substantial differences in the sequence in a helix 6 of the large subunit of these enzymes, which is intriguing since residues of this region had been previously shown to influence the ability of Ribulose bisphosphate carboxylase to discriminate between CO2 and O2, presumably by influencing the adjacent flexible loop 6 region. Sequence divergence at this and other key regions might contribute to the substantial differences in the substrate specificity factor of the chromophyte/rhodophyte enzyme. Initial studies on probing the basis for the high substrate specificity factor employed single amino acid substitutions in the recombinant cyanobacterial Ribulose bisphosphate carboxylase. Residues in the vicinity of loop 6 were changed to reflect the corresponding residues in the chromophyte/rhodophyte large subunit. Some changes in the substrate specificity factor were noted, as were alterations in other important kinetic parameters. Since marine algae show little evidence of photorespiratory metabolism, the high substrate specificity of Ribulose bisphosphate carboxylase is consistent with the physiology of these organisms. The results of this study provide further evidence that the properties of this enzyme may evolve or change according to the environment in which the host organism is found.
Mark R Harpel - One of the best experts on this subject based on the ideXlab platform.
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facilitation of the terminal proton transfer reaction of Ribulose 1 5 bisphosphate carboxylase oxygenase by active site lys166
Biochemistry, 1996Co-Authors: Mark R Harpel, Fred C HartmanAbstract:The terminal step in the carboxylation pathway catalyzed by Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is stereospecific protonation of the C-2 aci-acid of 3-phosphoglycerate (PGA). ...
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beta elimination of phosphate from reaction intermediates by site directed mutants of Ribulose bisphosphate carboxylase oxygenase
Journal of Biological Chemistry, 1994Co-Authors: Frank W Larimer, Mark R Harpel, Fred C HartmanAbstract:Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodospirillum rubrum Ribulose-bisphosphate carboxylase/oxygenase are positioned to serve as hydrogen-bond donors for the C1 phosphate of Ribulose bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Branden, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functional contributions of the residues implicated in Ribulose bisphosphate binding and intermediate stabilization, we have replaced them individually with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on enolization activity), increase of Km (Ribulose bisphosphate), and decrease of carboxylation: oxygenation specificity. During catalytic turnover of Ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, reflecting beta-elimination of phosphate from the enediol(ate) intermediate. This side product is not observed with wild-type enzyme, nor has it been reported with mutant enzymes characterized previously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the acicarbanion of 3-phosphoglycerate (the terminal intermediate). These data provide direct evidence that phosphate ligands stabilize conformations of intermediates that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.
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perturbation of reaction intermediate partitioning by a site directed mutant of Ribulose bisphosphate carboxylase oxygenase
Journal of Biological Chemistry, 1993Co-Authors: Eva H Lee, Mark R Harpel, Yuhru Chen, Fred C HartmanAbstract:Abstract To explore the roles of active-site Glu48 of Ribulose-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, the E48Q mutant has been characterized with respect to kinetics and product distribution. Although the kcat for carboxylase activity is only 0.6% of the wild-type value, the mutant retains full activity in catalyzing the conversion of the carboxylated reaction intermediate to 3-phosphoglycerate and retains 10% of the normal activity in catalyzing the enolization of Ribulose bisphosphate. Thus, the mutant is preferentially impaired in the carboxylation step. Partitioning of the enediol(ate) intermediate during turnover of Ribulose bisphosphate is perturbed dramatically in the case of the mutant protein. Whereas the wild-type enzyme displays a CO2/O2 specificity factor of 11, the corresponding parameter of the mutant is only 0.3, thereby signifying a shift of the relative reactivity of the enediol(ate) in favor of O2. The mutant protein is also unable to protect the enediol(ate) against misprotonation with consequential conversion of Ribulose bisphosphate to xylulose bisphosphate. This side reaction, undetected with wild-type R. rubrum enzyme, proceeds as rapidly as carboxylation of D-Ribulose 1,5-bisphosphate by the E48Q mutant. Formation of xylulose bisphosphate by the mutant does not appear to account for the decline in carboxylase activity that occurs during the course of an assay. These studies demonstrate the multiple functionalities of Glu48 in the facilitation of catalysis and in directing intermediate partitioning in the preferred direction.
