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Sherry L. Mowbray - One of the best experts on this subject based on the ideXlab platform.
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Structures of type B Ribose 5-Phosphate isomerase from Trypanosoma cruzi shed light on the determinants of sugar specificity in the structural family.
FEBS Journal, 2011Co-Authors: A.l. Stern, A. Naworyta, Juan José Cazzulo, Sherry L. MowbrayAbstract:Ribose-5-Phosphate isomerase (Rpi; EC 5.3.1.6) is a key activity of the pentose Phosphate pathway. Two unrelated types of sequence/structure possess this activity: type A Rpi (present in most organisms) and type B Rpi (RpiB) (in some bacteria and parasitic protozoa). In the present study, we report enzyme kinetics and crystallographic studies of the RpiB from the human pathogen, Trypanosoma cruzi. Structures of the wild-type and a Cys69Ala mutant enzyme, alone or bound to Phosphate, D-Ribose 5-Phosphate, or the inhibitors 4-phospho-D-erythronohydroxamic acid and D-allose 6-Phosphate, highlight features of the active site, and show that small conformational changes are linked to binding. Kinetic studies confirm that, similar to the RpiB from Mycobacterium tuberculosis, the T. cruzi enzyme can isomerize D-Ribose 5-Phosphate effectively, but not the 6-carbon sugar D-allose 6-Phosphate; instead, this sugar acts as an inhibitor of both enzymes. The behaviour is distinct from that of the more closely related (to T. cruzi RpiB) Escherichia coli enzyme, which can isomerize both types of sugars. The hypothesis that differences in a Phosphate-binding loop near the active site were linked to the differences in specificity was tested by construction of a mutant T. cruzi enzyme with a sequence in this loop more similar to that of E. coli RpiB; this mutant enzyme gained the ability to act on the 6-carbon sugar. The combined information allows us to distinguish the two types of specificity patterns in other available sequences. The results obtained in the present study provide insights into the action of RpiB enzymes generally, and also comprise a firm basis for future work in drug design.
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competitive inhibitors of type b Ribose 5 Phosphate isomerases design synthesis and kinetic evaluation of new d allose and d allulose 6 Phosphate derivatives
Carbohydrate Research, 2009Co-Authors: S Mariano, Sherry L. Mowbray, Annette K. Roos, Laurent SalmonAbstract:Abstract This study reports syntheses of d -allose 6-Phosphate (All6P), d -allulose (or d -psicose) 6-Phosphate (Allu6P), and seven d -Ribose 5-Phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichia coli (EcRpiB). 5-Phospho- d -ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho- d -ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho- d -ribonohydroxamic acid and N -(5-phospho- d -ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho- d -ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacterium tuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho- d -ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K i value of 9 μM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.
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d Ribose 5 Phosphate isomerase b from escherichia coli is also a functional d allose 6 Phosphate isomerase while the mycobacterium tuberculosis enzyme is not
Journal of Molecular Biology, 2008Co-Authors: Annette K. Roos, S Mariano, Laurent Salmon, Eva Kowalinski, Sherry L. MowbrayAbstract:Interconversion of D-Ribose-5-Phosphate (R5P) and D-ribulose-5-Phosphate is an important step in the pentose Phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-Phosphate (All6P) and D-allulose-6-Phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate Ribose-5-Phosphate, shed additional light on the reaction mechanism of RpiBs generally.
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Synthesis of 5-deoxy-5-phospho-d-ribonohydroxamic acid: a new competitive and selective inhibitor of type B Ribose-5-Phosphate isomerase from Mycobacterium tuberculosis
Tetrahedron Letters, 2005Co-Authors: Emmanuel S Burgos, Sherry L. Mowbray, Annette K. Roos, Laurent SalmonAbstract:Ribose 5-Phosphate isomerase (Rpi) is one of the major enzymes of the pentose Phosphate pathway, where it catalyses the inter-conversion of Ribose 5-Phosphate (R5P) and ribulose 5-Phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms Mycobacterium tuberculosis (Mt) and Escherichia coli (Ec) from a structural and functional point of view.Since the E. coli genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that EcRpiB has a different role as an allose-6-Phosphate isomerase. Activity measurements presented here show that EcRpiB does have this second activity. In the M. tuberculosis genome there is only a gene for RpiB. The crystal structure of MtRpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for MtRpiB and for other RpiBs in general. Activity measurements of MtRpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-Phosphate reaction. Differences observed in the active site between EcRpiB and MtRpiB explain these kinetic results. Activity measurements and a structure of an EcRpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. Inhibition studies have uncovered a compound that selectively inhibits MtRpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.
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Competitive Inhibitors of Mycobacterium tuberculosis Ribose-5-Phosphate Isomerase B Reveal New Information about the Reaction Mechanism
Journal of Biological Chemistry, 2004Co-Authors: Annette K. Roos, Laurent Salmon, Daniel J. Ericsson, Emmanuel S Burgos, Sherry L. MowbrayAbstract:Abstract Ribose-5-Phosphate isomerase (Rpi), an important enzyme in the pentose Phosphate pathway, catalyzes the interconversion of ribulose 5-Phosphate and Ribose 5-Phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (Km 57 μm) and 4-phospho-d-erythronate (Ki 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the Ribose 5-Phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.
Ulf Hanefeld - One of the best experts on this subject based on the ideXlab platform.
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Discovery and engineering of an aldehyde tolerant 2-deoxy-d-Ribose 5-Phosphate aldolase (Dera) from pectobacterium atrosepticum
Catalysts, 2020Co-Authors: Meera Haridas, Carolin Bisterfeld, Le Min Chen, Stefan R. Marsden, Fabio Tonin, Rosario Médici, Adolfo M. Iribarren, Elizabeth S. Lewkowicz, Peter-leon Hagedoorn, Ulf HanefeldAbstract:DERA (2-Deoxy-D-Ribose 5-Phosphate aldolase) is the only known aldolase that accepts two aldehyde substrates, which makes it an attractive catalyst for the synthesis of a chiral polyol motif that is present in several pharmaceuticals, such as atorvastatin and pravastatin. However, inactivation of the enzyme in the presence of aldehydes hinders its practical application. Whole cells of Pectobacterium atrosepticum were reported to exhibit good tolerance toward acetaldehyde and to afford 2-deoxyRibose 5-Phosphate with good yields. The DERA gene (PaDERA) was identified, and both the wild-type and a C49M mutant were heterologously expressed in Escherichia coli. The purification protocol was optimized and an initial biochemical characterization was conducted. Unlike other DERAs, which show a maximal activity between pH 4.0 and 7.5, PaDERA presented an optimum pH in the alkaline range between 8.0 and 9.0. This could warrant its use for specific syntheses in the future. PaDERA also displayed fourfold higher specific activity than DERA from E. coli (EcDERA) and displayed a promising acetaldehyde resistance outside the whole-cell environment. The C49M mutation, which was previously identified to increase acetaldehyde tolerance in EcDERA, also led to significant improvements in the acetaldehyde tolerance of PaDERA.
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2-Deoxy-D-Ribose-5-Phosphate aldolase (DERA): applications and modifications.
Applied Microbiology and Biotechnology, 2018Co-Authors: Meera Haridas, Eman M. M. Abdelraheem, Ulf HanefeldAbstract:© 2018, The Author(s). 2-Deoxy-d-Ribose-5-Phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.
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2 deoxy d Ribose 5 Phosphate aldolase dera applications and modifications
Applied Microbiology and Biotechnology, 2018Co-Authors: Meera Haridas, Eman M. M. Abdelraheem, Ulf HanefeldAbstract:2-Deoxy-d-Ribose-5-Phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.
Annette K. Roos - One of the best experts on this subject based on the ideXlab platform.
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competitive inhibitors of type b Ribose 5 Phosphate isomerases design synthesis and kinetic evaluation of new d allose and d allulose 6 Phosphate derivatives
Carbohydrate Research, 2009Co-Authors: S Mariano, Sherry L. Mowbray, Annette K. Roos, Laurent SalmonAbstract:Abstract This study reports syntheses of d -allose 6-Phosphate (All6P), d -allulose (or d -psicose) 6-Phosphate (Allu6P), and seven d -Ribose 5-Phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichia coli (EcRpiB). 5-Phospho- d -ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho- d -ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho- d -ribonohydroxamic acid and N -(5-phospho- d -ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho- d -ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacterium tuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho- d -ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K i value of 9 μM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.
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d Ribose 5 Phosphate isomerase b from escherichia coli is also a functional d allose 6 Phosphate isomerase while the mycobacterium tuberculosis enzyme is not
Journal of Molecular Biology, 2008Co-Authors: Annette K. Roos, S Mariano, Laurent Salmon, Eva Kowalinski, Sherry L. MowbrayAbstract:Interconversion of D-Ribose-5-Phosphate (R5P) and D-ribulose-5-Phosphate is an important step in the pentose Phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-Phosphate (All6P) and D-allulose-6-Phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate Ribose-5-Phosphate, shed additional light on the reaction mechanism of RpiBs generally.
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Synthesis of 5-deoxy-5-phospho-d-ribonohydroxamic acid: a new competitive and selective inhibitor of type B Ribose-5-Phosphate isomerase from Mycobacterium tuberculosis
Tetrahedron Letters, 2005Co-Authors: Emmanuel S Burgos, Sherry L. Mowbray, Annette K. Roos, Laurent SalmonAbstract:Ribose 5-Phosphate isomerase (Rpi) is one of the major enzymes of the pentose Phosphate pathway, where it catalyses the inter-conversion of Ribose 5-Phosphate (R5P) and ribulose 5-Phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms Mycobacterium tuberculosis (Mt) and Escherichia coli (Ec) from a structural and functional point of view.Since the E. coli genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that EcRpiB has a different role as an allose-6-Phosphate isomerase. Activity measurements presented here show that EcRpiB does have this second activity. In the M. tuberculosis genome there is only a gene for RpiB. The crystal structure of MtRpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for MtRpiB and for other RpiBs in general. Activity measurements of MtRpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-Phosphate reaction. Differences observed in the active site between EcRpiB and MtRpiB explain these kinetic results. Activity measurements and a structure of an EcRpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. Inhibition studies have uncovered a compound that selectively inhibits MtRpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.
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Competitive Inhibitors of Mycobacterium tuberculosis Ribose-5-Phosphate Isomerase B Reveal New Information about the Reaction Mechanism
Journal of Biological Chemistry, 2004Co-Authors: Annette K. Roos, Laurent Salmon, Daniel J. Ericsson, Emmanuel S Burgos, Sherry L. MowbrayAbstract:Abstract Ribose-5-Phosphate isomerase (Rpi), an important enzyme in the pentose Phosphate pathway, catalyzes the interconversion of ribulose 5-Phosphate and Ribose 5-Phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (Km 57 μm) and 4-phospho-d-erythronate (Ki 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the Ribose 5-Phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.
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Mycobacterium tuberculosis Ribose-5-Phosphate isomerase has a known fold, but a novel active site
Journal of Molecular Biology, 2004Co-Authors: Annette K. Roos, C. Evalena Andersson, Terese Bergfors, Micael Jacobsson, T. Alwyn Jones, Anders Karlen, Torsten Unge, Sherry L. MowbrayAbstract:Ribose-5-Phosphate isomerases (EC 5.3.1.6) inter-convert Ribose-5-Phosphate and ribulose-5-Phosphate. This reaction allows the synthesis of Ribose from other sugars, as well a means for salvage of carbohydrates after nucleotide breakdown. Two unrelated types of enzyme are known to catalyze the isomerization. The most common one, RpiA, is present in almost all organisms. The second type, RpiB, is found in many bacterial species.Here, we demonstrate that the RpiB from Mycobacterium tuberculosis (Rv2465c) has catalytic properties very similar to those previously reported for the Escherichia coli RpiB enzyme. Further, we report the structure of the mycobacterial enzyme, solved by molecular replacement and refined to 1.88A resolution. Comparison with the E.coli structure shows that there are important differences in the two active sites, including a change in the position and nature of the catalytic base. Sequence comparisons reveal that the M.tuberculosis and E.coli RpiB enzymes are in fact representative of two distinct sub-families. The mycobacterial enzyme represents a type found only in actinobacteria, while the enzyme from E.coli is typical of that seen in many other bacterial proteomes. Both RpiBs are very different from RpiA in structure as well as in the construction of the active site. Docking studies allow additional insights into the reactions of all three enzymes, and show that many features of the mechanism are preserved despite the different catalytic components.
Laurent Salmon - One of the best experts on this subject based on the ideXlab platform.
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competitive inhibitors of type b Ribose 5 Phosphate isomerases design synthesis and kinetic evaluation of new d allose and d allulose 6 Phosphate derivatives
Carbohydrate Research, 2009Co-Authors: S Mariano, Sherry L. Mowbray, Annette K. Roos, Laurent SalmonAbstract:Abstract This study reports syntheses of d -allose 6-Phosphate (All6P), d -allulose (or d -psicose) 6-Phosphate (Allu6P), and seven d -Ribose 5-Phosphate isomerase (Rpi) inhibitors. The inhibitors were designed as analogues of the 6-carbon high-energy intermediate postulated for the All6P to Allu6P isomerization reaction (Allpi activity) catalyzed by type B Rpi from Escherichia coli (EcRpiB). 5-Phospho- d -ribonate, easily obtained through oxidative cleavage of either All6P or Allu6P, led to the original synthon 5-dihydrogenophospho- d -ribono-1,4-lactone from which the other inhibitors could be synthesized through nucleophilic addition in one step. Kinetic evaluation on Allpi activity of EcRpiB shows that two of these compounds, 5-phospho- d -ribonohydroxamic acid and N -(5-phospho- d -ribonoyl)-methylamine, indeed behave as new efficient inhibitors of EcRpiB; further, 5-phospho- d -ribonohydroxamic acid was demonstrated to have competitive inhibition. Kinetic evaluation on Rpi activity of both EcRpiB and RpiB from Mycobacterium tuberculosis (MtRpiB) shows that several of the designed 6-carbon high-energy intermediate analogues are new competitive inhibitors of both RpiBs. One of them, 5-phospho- d -ribonate, not only appears as the strongest competitive inhibitor of a Rpi ever reported in the literature, with a K i value of 9 μM for MtRpiB, but also displays specific inhibition of MtRpiB versus EcRpiB.
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d Ribose 5 Phosphate isomerase b from escherichia coli is also a functional d allose 6 Phosphate isomerase while the mycobacterium tuberculosis enzyme is not
Journal of Molecular Biology, 2008Co-Authors: Annette K. Roos, S Mariano, Laurent Salmon, Eva Kowalinski, Sherry L. MowbrayAbstract:Interconversion of D-Ribose-5-Phosphate (R5P) and D-ribulose-5-Phosphate is an important step in the pentose Phosphate pathway. Two unrelated enzymes with R5P isomerase activity were first identified in Escherichia coli, RpiA and RpiB. In this organism, the essential 5-carbon sugars were thought to be processed by RpiA, while the primary role of RpiB was suggested to instead be interconversion of the rare 6-carbon sugars D-allose-6-Phosphate (All6P) and D-allulose-6-Phosphate. In Mycobacterium tuberculosis, where only an RpiB is found, the 5-carbon sugars are believed to be the enzyme's primary substrates. Here, we present kinetic studies examining the All6P isomerase activity of the RpiBs from these two organisms and show that only the E. coli enzyme can catalyze the reaction efficiently. All6P instead acts as an inhibitor of the M. tuberculosis enzyme in its action on R5P. X-ray studies of the M. tuberculosis enzyme co-crystallized with All6P and 5-deoxy-5-phospho-D-ribonohydroxamate (an inhibitor designed to mimic the 6-carbon sugar) and comparison with the E. coli enzyme's structure allowed us to identify differences in the active sites that explain the kinetic results. Two other structures, that of a mutant E. coli RpiB in which histidine 99 was changed to asparagine and that of wild-type M. tuberculosis enzyme, both co-crystallized with the substrate Ribose-5-Phosphate, shed additional light on the reaction mechanism of RpiBs generally.
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Ribose 5-Phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism.
Biochemical Journal, 2006Co-Authors: Ana L. Stern, Laurent Salmon, Emmanuel S Burgos, Juan José CazzuloAbstract:Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose Phosphate pathway, probably essential for protection against oxidative stress and also for R5P (Ribose 5-Phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (Ribose 5-Phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the Ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.
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Synthesis of 5-deoxy-5-phospho-d-ribonohydroxamic acid: a new competitive and selective inhibitor of type B Ribose-5-Phosphate isomerase from Mycobacterium tuberculosis
Tetrahedron Letters, 2005Co-Authors: Emmanuel S Burgos, Sherry L. Mowbray, Annette K. Roos, Laurent SalmonAbstract:Ribose 5-Phosphate isomerase (Rpi) is one of the major enzymes of the pentose Phosphate pathway, where it catalyses the inter-conversion of Ribose 5-Phosphate (R5P) and ribulose 5-Phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms Mycobacterium tuberculosis (Mt) and Escherichia coli (Ec) from a structural and functional point of view.Since the E. coli genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that EcRpiB has a different role as an allose-6-Phosphate isomerase. Activity measurements presented here show that EcRpiB does have this second activity. In the M. tuberculosis genome there is only a gene for RpiB. The crystal structure of MtRpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for MtRpiB and for other RpiBs in general. Activity measurements of MtRpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-Phosphate reaction. Differences observed in the active site between EcRpiB and MtRpiB explain these kinetic results. Activity measurements and a structure of an EcRpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. Inhibition studies have uncovered a compound that selectively inhibits MtRpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.
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Competitive Inhibitors of Mycobacterium tuberculosis Ribose-5-Phosphate Isomerase B Reveal New Information about the Reaction Mechanism
Journal of Biological Chemistry, 2004Co-Authors: Annette K. Roos, Laurent Salmon, Daniel J. Ericsson, Emmanuel S Burgos, Sherry L. MowbrayAbstract:Abstract Ribose-5-Phosphate isomerase (Rpi), an important enzyme in the pentose Phosphate pathway, catalyzes the interconversion of ribulose 5-Phosphate and Ribose 5-Phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (Km 57 μm) and 4-phospho-d-erythronate (Ki 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the Ribose 5-Phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.
Meera Haridas - One of the best experts on this subject based on the ideXlab platform.
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Discovery and engineering of an aldehyde tolerant 2-deoxy-d-Ribose 5-Phosphate aldolase (Dera) from pectobacterium atrosepticum
Catalysts, 2020Co-Authors: Meera Haridas, Carolin Bisterfeld, Le Min Chen, Stefan R. Marsden, Fabio Tonin, Rosario Médici, Adolfo M. Iribarren, Elizabeth S. Lewkowicz, Peter-leon Hagedoorn, Ulf HanefeldAbstract:DERA (2-Deoxy-D-Ribose 5-Phosphate aldolase) is the only known aldolase that accepts two aldehyde substrates, which makes it an attractive catalyst for the synthesis of a chiral polyol motif that is present in several pharmaceuticals, such as atorvastatin and pravastatin. However, inactivation of the enzyme in the presence of aldehydes hinders its practical application. Whole cells of Pectobacterium atrosepticum were reported to exhibit good tolerance toward acetaldehyde and to afford 2-deoxyRibose 5-Phosphate with good yields. The DERA gene (PaDERA) was identified, and both the wild-type and a C49M mutant were heterologously expressed in Escherichia coli. The purification protocol was optimized and an initial biochemical characterization was conducted. Unlike other DERAs, which show a maximal activity between pH 4.0 and 7.5, PaDERA presented an optimum pH in the alkaline range between 8.0 and 9.0. This could warrant its use for specific syntheses in the future. PaDERA also displayed fourfold higher specific activity than DERA from E. coli (EcDERA) and displayed a promising acetaldehyde resistance outside the whole-cell environment. The C49M mutation, which was previously identified to increase acetaldehyde tolerance in EcDERA, also led to significant improvements in the acetaldehyde tolerance of PaDERA.
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2-Deoxy-D-Ribose-5-Phosphate aldolase (DERA): applications and modifications.
Applied Microbiology and Biotechnology, 2018Co-Authors: Meera Haridas, Eman M. M. Abdelraheem, Ulf HanefeldAbstract:© 2018, The Author(s). 2-Deoxy-d-Ribose-5-Phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.
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2 deoxy d Ribose 5 Phosphate aldolase dera applications and modifications
Applied Microbiology and Biotechnology, 2018Co-Authors: Meera Haridas, Eman M. M. Abdelraheem, Ulf HanefeldAbstract:2-Deoxy-d-Ribose-5-Phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.
