The Experts below are selected from a list of 135 Experts worldwide ranked by ideXlab platform
Jon S. Thorson - One of the best experts on this subject based on the ideXlab platform.
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Recombinant E. coli Prototype Strains for in Vivo Glycorandomization
ACS chemical biology, 2010Co-Authors: Gavin J. Williams, Jie Yang, Changsheng Zhang, Jon S. ThorsonAbstract:In vitro Glycorandomization is a powerful strategy to alter the glycosylation patterns of natural products and small molecule therapeutics. Yet, such in vitro methods are often difficult to scale and can be costly given the requirement to provide various nucleotides and cofactors. Here, we report the construction of several recombinant E. coli prototype strains that allow the facile production of a range of small molecule glycosides. This strategy relies on the engineered promiscuity of three key enzymes, an anomeric kinase, a sugar-1-phosphate nucleotidyltransferase, and a glycosyltransferase, as well as the ability of diverse small molecules to freely enter E. coli. Subsequently, this work is the first demonstration of “in vivo Glycorandomization” and offers vast combinatorial potential by simple fermentation.
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probing the aglycon promiscuity of an engineered glycosyltransferase
Angewandte Chemie, 2008Co-Authors: Richard W Gantt, Gavin J. Williams, Randal D Goff, Jon S. ThorsonAbstract:Sugars appended to pharmaceutically important natural products influence key pharmacological properties and/or molecular mechanism of action.[1] However, studies designed to systematically understand and/or exploit the role of carbohydrates in drug discovery are often limited by the availability of practical synthetic and/or biosynthetic tools.[2] Among the contemporary options to address this limitation,[3–4] chemoenzymatic Glycorandomization utilizes a set of flexible enzymes consisting of an anomeric kinase, sugar-1-phosphate nucleotidylytransferase, and natural product glycosyltransferase (GT).[4–6] While chemoenzymatic Glycorandomization has been successfully applied to alter the natural sugar moieties of numerous natural products,[4–8] the process remains primarily restricted by enzyme specificity and availability of suitable GTs for the target of interest. Thus, although there is precedent for improving non-glycosylated therapeutics via glycoconjugation, including colchicine,[9] mitomycin,[10] podophyllotoxin,[11] rapamycin,[12] isophosphoramide mustards,[13] or taxol,[14] such targets remain beyond chemoenzymatic strategies. Recent studies on OleD, the oleandomycin (1) GT from Streptomyces antibioticus (Scheme 1a), revealed an enhanced triple mutant (A242V/S132F/P67T, referred to herein as ‘ASP’) that displayed marked improvement in proficiency and substrate promiscuity.[4] To probe the synthetic utility of this enhanced catalyst and expand upon previous reports of acceptor promiscuity for wild-type (WT) OleD,[15] we report a comparison of the aglycon specificities of the WT and ‘ASP’ OleD variants toward 137 drug-like acceptors. This study highlights the ability of OleD variants to glucosylate a total of 71 diverse acceptors, catalyze iterative glycosylation with numerous substrates, and establishes OleD as the first multifunctional GT capable of generating O-, S- and N-glycosides.
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optimizing glycosyltransferase specificity via hot spot saturation mutagenesis presents a catalyst for novobiocin Glycorandomization
Chemistry & Biology, 2008Co-Authors: Gavin J. Williams, Changsheng Zhang, Randal D Goff, Jon S. ThorsonAbstract:A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.
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expanding the promiscuity of a natural product glycosyltransferase by directed evolution
Nature Chemical Biology, 2007Co-Authors: Gavin J. Williams, Changsheng Zhang, Jon S. ThorsonAbstract:Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development1. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels2, and/or even the molecular mechanism of action3. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the Glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.
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Selective detection of sugar phosphates by capillary electrophoresis/mass spectrometry and its application to an engineered E. coli host.
Chembiochem : a European journal of chemical biology, 2007Co-Authors: Joseph P. M. Hui, Jie Yang, Jon S. Thorson, Evelyn C. SooAbstract:A highly selective method employing capillary electrophoresis and electrospray mass spectrometry (CE-ESMS) with precursor ion scanning for fragment ions characteristic of phosphate-linked sugars was developed for the determination of "unnatural" sugar phosphates generated in vivo, as part of a natural product Glycorandomization study. Cell lysates from an engineered E. coli host were probed for "natural" and "unnatural" sugar phosphates resulting from in vivo galactokinase (GalK) bioconversions, and tandem mass spectrometry experiments were performed to confirm the identities of the sugar phosphates. Among the 22 cell lysates that were studied, 13 were found to contain the expected natural and "unnatural" sugar phosphates. This was in agreement with the GalK in vitro conversion yields, in which an in vitro yield of
Jie Yang - One of the best experts on this subject based on the ideXlab platform.
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Recombinant E. coli Prototype Strains for in Vivo Glycorandomization
ACS chemical biology, 2010Co-Authors: Gavin J. Williams, Jie Yang, Changsheng Zhang, Jon S. ThorsonAbstract:In vitro Glycorandomization is a powerful strategy to alter the glycosylation patterns of natural products and small molecule therapeutics. Yet, such in vitro methods are often difficult to scale and can be costly given the requirement to provide various nucleotides and cofactors. Here, we report the construction of several recombinant E. coli prototype strains that allow the facile production of a range of small molecule glycosides. This strategy relies on the engineered promiscuity of three key enzymes, an anomeric kinase, a sugar-1-phosphate nucleotidyltransferase, and a glycosyltransferase, as well as the ability of diverse small molecules to freely enter E. coli. Subsequently, this work is the first demonstration of “in vivo Glycorandomization” and offers vast combinatorial potential by simple fermentation.
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Selective detection of sugar phosphates by capillary electrophoresis/mass spectrometry and its application to an engineered E. coli host.
Chembiochem : a European journal of chemical biology, 2007Co-Authors: Joseph P. M. Hui, Jie Yang, Jon S. Thorson, Evelyn C. SooAbstract:A highly selective method employing capillary electrophoresis and electrospray mass spectrometry (CE-ESMS) with precursor ion scanning for fragment ions characteristic of phosphate-linked sugars was developed for the determination of "unnatural" sugar phosphates generated in vivo, as part of a natural product Glycorandomization study. Cell lysates from an engineered E. coli host were probed for "natural" and "unnatural" sugar phosphates resulting from in vivo galactokinase (GalK) bioconversions, and tandem mass spectrometry experiments were performed to confirm the identities of the sugar phosphates. Among the 22 cell lysates that were studied, 13 were found to contain the expected natural and "unnatural" sugar phosphates. This was in agreement with the GalK in vitro conversion yields, in which an in vitro yield of
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Structure-based engineering of E. coli galactokinase as a first step toward in vivo Glycorandomization.
Chemistry & biology, 2005Co-Authors: Jie Yang, Lesley Liu, Jianchun Liao, Jon S. ThorsonAbstract:Summary In vitro Glycorandomization is a rapid chemoenzymatic strategy to diversify complex natural product scaffolds. The Glycorandomization sugar activation pathway is dependent upon the efficient construction of diverse sugar-1-phosphate libraries. In the context of the previously evolved GalK Y371H "gatekeeper" mutation, the active site M173L mutation described herein presents a kinase with remarkably broadened substrate range to include 28 diverse natural and unnatural sugars. Among these new substrates, 6-azido-6-deoxy-galactose and 6-azido-6-deoxy-glucose present unique chemical probes to assess the utility of an E. coli Y371H/M173L-GalK-overproducing strain to generate unnatural sugar-1-phosphates in vivo. Remarkably, the in vivo conversion of both unnatural sugars rival that demonstrated in vitro. This notable in vivo success stands as the first step toward constructing short sugar-activation pathways in vivo and, ultimately, in vivo natural-product Glycorandomization.
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Natural product Glycorandomization.
Bioorganic & medicinal chemistry, 2004Co-Authors: Jie Yang, Dirk Hoffmeister, Lesley Liu, Jon S. ThorsonAbstract:Glycorandomization is a chemoenzymatic strategy that overcomes the limitations in natural product derivatization associated with both solely chemistry-based approaches or in vivo engineering. In this article we present the basic strategies for Glycorandomization development as a next-generation tool in drug discovery.
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Creation of the first anomeric d/l-sugar kinase by means of directed evolution
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Dirk Hoffmeister, Jie Yang, Lesley Liu, Jon S. ThorsonAbstract:Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as d-galacturonic acid, d-talose, l-altrose, and l-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product Glycorandomization.
Jiqing Jiang - One of the best experts on this subject based on the ideXlab platform.
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Antibiotic optimization via in vitro Glycorandomization
Nature Biotechnology, 2003Co-Authors: Christoph Albermann, Jiqing Jiang, Jianchun Liao, Changsheng Zhang, Jon S. ThorsonAbstract:In nature, the attachment of sugars to small molecules is often used to mediate targeting, mechanism of action and/or pharmacology. As an alternative to pathway engineering or total synthesis, we report a useful method, in vitro Glycorandomization (IVG), to diversify the glycosylation patterns of complex natural products. We have used flexible glycosyltransferases on nucleotide diphosphosugar (NDP-sugar) libraries to generate glycorandomized natural products and then applied chemoselective ligation to produce monoglycosylated vancomycins that rival vancomycin.
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Studies on the substrate specificity of Escherichia coli galactokinase.
Organic letters, 2003Co-Authors: Jie Yang, John B. Biggins, Jiqing Jiang, Qiang Jia, Jie Shen, Jingjing Zhao, Joshua J. Schmidt, Peng George Wang, Jon S. ThorsonAbstract:In vitro Glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes.
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substrate specificity of novm implications for novobiocin biosynthesis and Glycorandomization
Organic Letters, 2003Co-Authors: Christoph Albermann, William A. Barton, John B. Biggins, Jiqing Jiang, Dimitar B. Nikolov, Aileen Soriano, Heidi R Vollmer, Jacob Lesniak, Jon S. ThorsonAbstract:In an effort to expand the scope of natural product in vitro Glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new “unnatural” natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.
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Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: William A. Barton, Jon S. Thorson, John B. Biggins, Jiqing Jiang, Dimitar B. NikolovAbstract:In vitro “Glycorandomization” is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for Glycorandomization, we now report a structure-based engineering approach to increase the diversity of α-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 α-d-glucopyranosyl phosphate thymidylyltransferase (Ep). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three “designed” mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of α-d-hexopyranosyl phosphates accepted by Ep to include α-d-allo-, α-d-altro-, and α-d-talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro Glycorandomization.
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Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward Glycorandomization
Nature Structural Biology, 2001Co-Authors: William A. Barton, Jon S. Thorson, John B. Biggins, Jiqing Jiang, Jacob Lesniak, Philip D. Jeffrey, K. R. Rajashankar, Dimitar B. NikolovAbstract:Metabolite glycosylation is affected by three classes of enzymes: nucleotidylyltransferases, which activate sugars as nucleotide diphospho-derivatives, intermediate sugar-modifying enzymes and glycosyltransferases, which transfer the final derivatized activated sugars to aglycon substrates. One of the first crystal structures of an enzyme responsible for the first step in this cascade, α-D-glucopyranosyl phosphate thymidylyltransferase (E_p) from Salmonella , in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 Å and 2.1 Å resolution, respectively. These structures, in conjunction with the kinetic characterization of E_p, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of E_p produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type E_p. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral component of in vitro glycosylation systems developed for the production of diverse glycorandomized libraries.
Christoph Albermann - One of the best experts on this subject based on the ideXlab platform.
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Diversifying vancomycin via chemoenzymatic strategies.
Organic letters, 2005Co-Authors: Christoph Albermann, And Changsheng Zhang, Jon S. ThorsonAbstract:The rapid diversification of glycopeptides via Glycorandomization reveals that significantly diverse substitutions are tolerated and suggests there may be a synergistic benefit to the construction of mechanistically related natural product core scaffold fusions. This work also further highlights the utility of chemoenzymatic approaches to diversify complex natural product architectures.
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Antibiotic optimization via in vitro Glycorandomization
Nature Biotechnology, 2003Co-Authors: Christoph Albermann, Jiqing Jiang, Jianchun Liao, Changsheng Zhang, Jon S. ThorsonAbstract:In nature, the attachment of sugars to small molecules is often used to mediate targeting, mechanism of action and/or pharmacology. As an alternative to pathway engineering or total synthesis, we report a useful method, in vitro Glycorandomization (IVG), to diversify the glycosylation patterns of complex natural products. We have used flexible glycosyltransferases on nucleotide diphosphosugar (NDP-sugar) libraries to generate glycorandomized natural products and then applied chemoselective ligation to produce monoglycosylated vancomycins that rival vancomycin.
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substrate specificity of novm implications for novobiocin biosynthesis and Glycorandomization
Organic Letters, 2003Co-Authors: Christoph Albermann, William A. Barton, John B. Biggins, Jiqing Jiang, Dimitar B. Nikolov, Aileen Soriano, Heidi R Vollmer, Jacob Lesniak, Jon S. ThorsonAbstract:In an effort to expand the scope of natural product in vitro Glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new “unnatural” natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.
Changsheng Zhang - One of the best experts on this subject based on the ideXlab platform.
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Recombinant E. coli Prototype Strains for in Vivo Glycorandomization
ACS chemical biology, 2010Co-Authors: Gavin J. Williams, Jie Yang, Changsheng Zhang, Jon S. ThorsonAbstract:In vitro Glycorandomization is a powerful strategy to alter the glycosylation patterns of natural products and small molecule therapeutics. Yet, such in vitro methods are often difficult to scale and can be costly given the requirement to provide various nucleotides and cofactors. Here, we report the construction of several recombinant E. coli prototype strains that allow the facile production of a range of small molecule glycosides. This strategy relies on the engineered promiscuity of three key enzymes, an anomeric kinase, a sugar-1-phosphate nucleotidyltransferase, and a glycosyltransferase, as well as the ability of diverse small molecules to freely enter E. coli. Subsequently, this work is the first demonstration of “in vivo Glycorandomization” and offers vast combinatorial potential by simple fermentation.
-
optimizing glycosyltransferase specificity via hot spot saturation mutagenesis presents a catalyst for novobiocin Glycorandomization
Chemistry & Biology, 2008Co-Authors: Gavin J. Williams, Changsheng Zhang, Randal D Goff, Jon S. ThorsonAbstract:A comprehensive two-phase "hot spot" saturation mutagenesis strategy for the rapid evolution of glycosyltransferase (GT) specificity for nonnatural acceptors is described. Specifically, the application of a high-throughput screen (based on the fluorescent acceptor umbelliferone) was used to identify key amino acid hot spots that contribute to GT proficiency and/or promiscuity. Saturation mutagenesis of the corresponding hot spots facilitated the utilization of a lower-throughput screen to provide OleD prodigy capable of efficiently glycosylating the nonnatural acceptor novobiocic acid with an array of unique sugars. Incredibly, even in the absence of a high-throughput screen for novobiocic acid glycosylation, this approach rapidly led to improvements in the desired catalytic activity of several hundred-fold.
-
expanding the promiscuity of a natural product glycosyltransferase by directed evolution
Nature Chemical Biology, 2007Co-Authors: Gavin J. Williams, Changsheng Zhang, Jon S. ThorsonAbstract:Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development1. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels2, and/or even the molecular mechanism of action3. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the Glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.
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Antibiotic optimization via in vitro Glycorandomization
Nature Biotechnology, 2003Co-Authors: Christoph Albermann, Jiqing Jiang, Jianchun Liao, Changsheng Zhang, Jon S. ThorsonAbstract:In nature, the attachment of sugars to small molecules is often used to mediate targeting, mechanism of action and/or pharmacology. As an alternative to pathway engineering or total synthesis, we report a useful method, in vitro Glycorandomization (IVG), to diversify the glycosylation patterns of complex natural products. We have used flexible glycosyltransferases on nucleotide diphosphosugar (NDP-sugar) libraries to generate glycorandomized natural products and then applied chemoselective ligation to produce monoglycosylated vancomycins that rival vancomycin.