The Experts below are selected from a list of 159 Experts worldwide ranked by ideXlab platform
Neil C Bruce - One of the best experts on this subject based on the ideXlab platform.
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cofactor regeneration by a soluble Pyridine Nucleotide transhydrogenase for biological production of hydromorphone
Applied and Environmental Microbiology, 2000Co-Authors: Birgitte Boonstra, Deborah A Rathbone, Christopher E French, Edward H Walker, Neil C BruceAbstract:We have applied the soluble Pyridine Nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP+-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble Pyridine Nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble Pyridine Nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble Pyridine Nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble Pyridine Nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.
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the udha gene of escherichia coli encodes a soluble Pyridine Nucleotide transhydrogenase
Journal of Bacteriology, 1999Co-Authors: Birgitte Boonstra, Christopher E French, Ian Wainwright, Neil C BruceAbstract:The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble Pyridine Nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a diNucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound Pyridine Nucleotide transhydrogenase.
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cloning sequence and properties of the soluble Pyridine Nucleotide transhydrogenase of pseudomonas fluorescens
Journal of Bacteriology, 1997Co-Authors: Christopher E French, Birgitte Boonstra, K A J Bufton, Neil C BruceAbstract:The gene encoding the soluble Pyridine Nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.
Birgitte Boonstra - One of the best experts on this subject based on the ideXlab platform.
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cofactor regeneration by a soluble Pyridine Nucleotide transhydrogenase for biological production of hydromorphone
Applied and Environmental Microbiology, 2000Co-Authors: Birgitte Boonstra, Deborah A Rathbone, Christopher E French, Edward H Walker, Neil C BruceAbstract:We have applied the soluble Pyridine Nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP+-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble Pyridine Nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble Pyridine Nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble Pyridine Nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble Pyridine Nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.
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the udha gene of escherichia coli encodes a soluble Pyridine Nucleotide transhydrogenase
Journal of Bacteriology, 1999Co-Authors: Birgitte Boonstra, Christopher E French, Ian Wainwright, Neil C BruceAbstract:The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble Pyridine Nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a diNucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound Pyridine Nucleotide transhydrogenase.
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cloning sequence and properties of the soluble Pyridine Nucleotide transhydrogenase of pseudomonas fluorescens
Journal of Bacteriology, 1997Co-Authors: Christopher E French, Birgitte Boonstra, K A J Bufton, Neil C BruceAbstract:The gene encoding the soluble Pyridine Nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.
Philip D Bragg - One of the best experts on this subject based on the ideXlab platform.
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involvement of histidine 91 of the beta subunit in proton translocation by the Pyridine Nucleotide transhydrogenase of escherichia coli
Biochemistry, 1995Co-Authors: Natalie A Glavas, Philip D BraggAbstract:: The Pyridine Nucleotide transhydrogenase (EC 1.6.1.1) carries out transmembrane proton translocation coupled to transfer of a hydride equivalent between NAD+ and NADP+. Mutations were made in histidine-91 of the beta subunit of the Pyridine Nucleotide transhydrogenase of Escherichia coli. This amino acid is the only conserved charged residue in the transmembrane domains of this enzyme and thus potentially is involved in proton translocation by the transhydrogenase. The mutant beta H91N retained 80% of the hydride transfer activity while proton translocation was reduced to 7%. This behavior is consistent with a role for beta His91 in the proton translocation pathway. Other mutations at this residue affected the conformation of the enzyme. Thus, the enzyme in mutants beta H91C, beta H91T, and beta H91S was unable to undergo the conformational change that occurred on binding of the substrates NADP+ or NADPH. By contrast, the enzyme in the beta H91K mutant was present in the NADP(H)-induced conformation even in the absence of these substrates. Further evidence for the linkage between beta His91 and the conformation of the beta subunit was obtained by labeling the transmembrane domain of the beta subunit with [14C]N,N'-dicyclohexylcarbodiimide (DCCD). Labeling occurred most readily with the enzyme of beta H91K. It is concluded that beta His91 is a component of the proton translocation pathway of the transhydrogenase and that its state of protonation is probably linked to conformational changes induced by binding/debinding of substrates during the catalytic cycle of the enzyme.
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topological analysis of the Pyridine Nucleotide transhydrogenase of escherichia coli using proteolytic enzymes
Biochimica et Biophysica Acta, 1991Co-Authors: Raymond C W Tong, Natalie A Glavas, Philip D BraggAbstract:The Pyridine Nucleotide transhydrogenase of Escherichia coli has an α 2 β 2 structure (α: M r , 54 000; β: M r , 48 700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the α subunit and the N-terminal 20–25 kDa region of the β subunit are composed of transmembranous α-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the α subunit. The β subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both α and β subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The β subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the β subunit. Digestion of the β subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the α and β subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the α subunit and the C-terminal 30 kDa region of the β subunit are exposed on the cytoplasmic side of the inner membrane of E. coli . Binding sites for Pyridine Nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.
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crosslinking and radiation inactivation analysis of the subunit structure of the Pyridine Nucleotide transhydrogenase of escherichia coli
Biochimica et Biophysica Acta, 1990Co-Authors: Michel Potier, Philip D BraggAbstract:Abstract The Pyridine Nucleotide transhydrogenase of Escherichia coli consists of two types of subunit (α: Mr 53 906; β: Mr 48 667). The purified and membrane-bound enzymes were crosslinked with a series of bifunctional crosslinking agents and by catalyzing the formation of inter-chain disulfides in the presence of cupric 1,10-phenanthrolinate. Crosslinked dimers α2, αβ and β2, and the trimer α2β were obtained. A small amount of tetramer, probably α2β2, was also formed. Radiation inactivation was used to determine the molecular size of the transhydrogenase. The radiation inactivation size (217 000) and chemical crosslinking are consistent with the structure (Mr 205 146) being the oligomer that is responsible for biological activity.
Christopher E French - One of the best experts on this subject based on the ideXlab platform.
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cofactor regeneration by a soluble Pyridine Nucleotide transhydrogenase for biological production of hydromorphone
Applied and Environmental Microbiology, 2000Co-Authors: Birgitte Boonstra, Deborah A Rathbone, Christopher E French, Edward H Walker, Neil C BruceAbstract:We have applied the soluble Pyridine Nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP+-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble Pyridine Nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble Pyridine Nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble Pyridine Nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble Pyridine Nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.
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the udha gene of escherichia coli encodes a soluble Pyridine Nucleotide transhydrogenase
Journal of Bacteriology, 1999Co-Authors: Birgitte Boonstra, Christopher E French, Ian Wainwright, Neil C BruceAbstract:The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble Pyridine Nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a diNucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound Pyridine Nucleotide transhydrogenase.
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cloning sequence and properties of the soluble Pyridine Nucleotide transhydrogenase of pseudomonas fluorescens
Journal of Bacteriology, 1997Co-Authors: Christopher E French, Birgitte Boonstra, K A J Bufton, Neil C BruceAbstract:The gene encoding the soluble Pyridine Nucleotide transhydrogenase (STH) of Pseudomonas fluorescens was cloned and expressed in Escherichia coli. STH is related to the flavoprotein disulfide oxidoreductases but lacks one of the conserved redox-active cysteine residues. The gene is highly similar to an E. coli gene of unknown function.
Charles H Williams - One of the best experts on this subject based on the ideXlab platform.
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effect of Pyridine Nucleotide on the oxidative half reaction of escherichia coli thioredoxin reductase
Biochemistry, 1995Co-Authors: Brett W Lennon, Charles H WilliamsAbstract:: The kinetics of the oxidative half-reaction between reduced thioredoxin reductase and oxidized thioredoxin measured in the presence and absence of Pyridine Nucleotide show a significant difference in the rates of the main phase of oxidation. When 1 equiv of NADPH is used to partially reduce the enzyme at pH 7.0 or 7.6, the observed rate of the catalytically competent phase of oxidation is essentially equal to kcat at that pH. This is about 50% of the rate of oxidation observed with enzyme fully reduced or partially reduced by the xanthine/xanthine oxidase system or by dithionite. Through the use of the nonreducible analog 3-aminoPyridine adenine diNucleotide phosphate we have shown that this decrease in observed rate of oxidation is linked to the concentration of Pyridine Nucleotide present. This suggests that the complexation of Pyridine Nucleotides with reduced thioredoxin reductase is able to effect a change in the rate-limiting steps of the oxidation of the enzyme by thioredoxin. This is the case even when substoichiometric quantities of 3-aminoPyridine adenine diNucleotide phosphate are present, which predicts that the binding to reduced enzyme is very tight. It is clear that the presence of 1 equiv of NADP+ is sufficient to cause the observed rate for the catalytically competent phase of oxidation to decrease to kcat. Thus, there is compelling evidence for a ternary complex mechanism for thioredoxin reductase.
