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Jon S Thorson

Publications and source records attributed to Jon S Thorson.

At least 19 recordsLinked to original sources
PubMed2006

The in vitro characterization of the iterative avermectin glycosyltransferase AveBI reveals reaction reversibility and sugar nucleotide flexibility.

The glycosyltransferase AveBI, which is involved in the biosynthesis of the macrolide antihelmintic avermectin (AVM), was characterized in vitro. AveBI was confirmed to catalyze two separate iterative additions of l-oleandrose, and the reversibility of AveBI-catalyzed reaction was also demonstrated. Investigation of sugar nucleotide specificity revealed 10 unique sugar nucleotide substrates which, in combination with five distinct aglycones, led to the production of 50 differentially glycosylated AVM variants.

Catalysis↗
PubMed2006

Colchicine glycorandomization influences cytotoxicity and mechanism of action.

The reaction of 70 unprotected, diversely functionalized free reducing sugars with methoxyamine-appended colchicine led to the production of a 58-member glycorandomized library. High-throughput cytotoxicity assays revealed glycosylation to modulate specificity and potency. Library members were also identified which, unlike the parent natural product (a destabilizer), stabilized in vitro tubulin polymerization in a manner similar to taxol. This study highlights a simple extension of neoglycorandomization toward amine-bearing scaffolds and the potential benefit of glycosylating nonglycosylated natural products.

Antineoplastic Agents↗
PubMed2006

Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions.

Glycosyltransferases (GTs), an essential class of ubiquitous enzymes, are generally perceived as unidirectional catalysts. In contrast, we report that four glycosyltransferases from two distinct natural product biosynthetic pathways-calicheamicin and vancomycin-readily catalyze reversible reactions, allowing sugars and aglycons to be exchanged with ease. As proof of the broader applicability of these new reactions, more than 70 differentially glycosylated calicheamicin and vancomycin variants are reported. This study suggests the reversibility of GT-catalyzed reactions may be general and useful for generating exotic nucleotide sugars, establishing in vitro GT activity in complex systems, and enhancing natural product diversity.

Aminoglycosides↗
PubMed2006

Structural insight into the self-sacrifice mechanism of enediyne resistance.

The recent discovery of the first "self-sacrifice" mechanism for bacterial resistance to the enediyne antitumor antibiotics, where enediyne-induced proteolysis of the resistance protein CalC inactivates both the highly reactive metabolite and the resistance protein, revealed yet another ingenious bacterial mechanism for controlling reactive metabolites. As reported herein, the first 3D structures of CalC and CalC in complex with calicheamicin (CLM) divulge CalC to be a member of the steroidogenic acute regulatory protein (StAR)-related transfer (START) domain superfamily. In contrast to previous studies of proteins known to bind DNA-damaging natural products ( e.g ., bleomycins, mitomycins, and nine-membered chromoprotein enediynes), this is the first demonstrated involvement of a START domain fold. Consistent with the CalC self-sacrifice mechanism, CLM in complex with CalC is positioned for direct hydrogen abstraction from Gly113 to initiate the oxidative proteolysis-based resistance mechanism. These structural studies also illuminate, for the first time, a small DNA-binding region within CalC that may serve to localize CalC to the enediyne target (DNA). Given the role of START domains in nuclear/cytosolic transport and translocation, this structural study also may implicate START domains as post-endocytotic intracellular chaperones for enediyne-based therapeutics such as MyloTarg.

Aminoglycosides↗
PubMed2006

Enzymatic tools for engineering natural product glycosylation.

Glycosylated natural products have served as reliable platforms for the development of many existing front-line drugs. In an effort to explore the contribution of the sugar constituents of these compounds, research groups have focused upon the development of chemical and enzymatic tools to diversify natural product glycosylation. Among the complementary routes available, in vivo pathway engineering, also referred to as 'combinatorial biosynthesis', is an emerging method that relies upon the co-expression of sugar biosynthetic gene cassettes and glycosyltransferases in a host organism to generate novel glycosylated natural products. An overview of recent progress in combinatorial biosynthesis is highlighted in this review, emphasizing the elucidation of nucleotide-sugar biosynthetic pathways and recent developments on glycosyltransferases.

Biological Products↗
PubMed2006

Natural product diversification using a non-natural cofactor analogue of S-adenosyl-L-methionine.

Adenosine analogues bearing either 5'-aziridine or 5'-N-mustard electrophiles are methyltransferase-dependent DNA alkylating agents. We present here a novel synthetic cofactor bearing a pendant 5'-amino acid N-mustard. Unlike previously studied synthetic cofactors, this material is very efficiently used by the natural product biosynthetic enzyme rebeccamycin methyltransferase (RebM) to generate a number of new rebeccamycin analogues. These data promote the notion that natural product methyltransferases can be used with non-natural cofactors to enhance the molecular diversity of natural product analogues for drug discovery. To our knowledge, this is the first documentation of a biological methyltransferase, other than DNA methyltransferases, that can exploit such synthetic cofactors.

Alkylation↗
PubMed2006

RebG- and RebM-catalyzed indolocarbazole diversification.

Rebeccamycin and staurosporine represent two broad classes of indolocarbazole glycoside natural products with antitumor properties. Based upon previous sequence annotation and in vivo studies, rebG encodes for the rebeccamycin N-glucosyltransferase, and rebM for the requisite 4'-O-methyltransferase. In the current study, an efficient in vivo biotransformation system for RebG was established in both Streptomyces lividans and Escherichia coli. Bioconversion experiments revealed RebG to glucosylate a set of indolocarbazole surrogates, the products of which could be further modified by in vitro RebM-catalyzed 4'-O-methylation. Both RebG and RebM displayed substrate promiscuity, and evidence for a remarkable lack of RebG regioselectivity in the presence of asymmetric substrates is also provided. In the context of the created indolocarbazole analogues, cytotoxicity assays also highlight the importance of 4'-O-methylation for their biological activity.

Bacterial Proteins↗
PubMed2006

Deciphering indolocarbazole and enediyne aminodideoxypentose biosynthesis through comparative genomics: insights from the AT2433 biosynthetic locus.

AT2433, an indolocarbazole antitumor antibiotic, is structurally distinguished by its aminodideoxypentose-containing disaccharide and asymmetrically halogenated N-methylated aglycon. Cloning and sequence analysis of AT2433 gene cluster and comparison of this locus with that encoding for rebeccamycin and the gene cluster encoding calicheamicin present an opportunity to study the aminodideoxypentose biosynthesis via comparative genomics. The locus was confirmed via in vitro biochemical characterization of two methyltransferases--one common to AT2433 and rebeccamycin, the other unique to AT2433--as well as via heterologous expression and in vivo bioconversion experiments using the AT2433 N-glycosyltransferase. Preliminary studies of substrate tolerance for these three enzymes reveal the potential to expand upon the enzymatic diversification of indolocarbazoles. Moreover, this work sets the stage for future studies regarding the origins of the indolocarbazole maleimide nitrogen and indolocarbazole asymmetry.

Amino Sugars↗
PubMed2006

A continuous assay for DNA cleavage using molecular break lights.

Exploring the properties of molecules that cleave DNA (i.e., enzymatic nucleases, chemical footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both laboratory and clinical applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, we describe the molecular break light assay, wherein a single oligo-nucleotide modified by a 5'-fluorophore-3'-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodology is highly convenient in that it is readily adaptable to common laboratory fluorometers and multi-well plate/ array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or "unnatural" transcription factor protection assay systems.

DNA↗
PubMed2005

'Sweetening' natural products via glycorandomization.

In an effort to explore the contribution of the sugar constituents of pharmaceutically relevant glycosylated natural products, chemists have developed glycosylation methods for the generation of 'glycorandomized' libraries. Each member of these libraries is uniquely differentiated by an attached carbohydrate. Recently, two complementary glycorandomization strategies have emerged: chemoenzymatic glycorandomization, a biocatalytic approach dependent upon the substrate promiscuity of enzymes to activate and attach sugars to natural products, and neoglycorandomization, an efficient one-step chemical sugar ligation reaction that does not require prior sugar protection or activation. These strategies are likely to have a significant impact on fundamental glycoscience and drug discovery.

Carbohydrate Conformation↗
PubMed2005

Structure, activity, synthesis and biosynthesis of aryl-C-glycosides.

The focus of this review is to highlight the structure, bioactivity and biosynthesis of naturally occurring aryl-C-glycosides. General synthetic methods and their relevance to proposed biochemical mechanisms for the aryl-C-glycoside bond formation are also presented.

Biological Products↗
PubMed2005

Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization.

Glycosylated natural products are reliable platforms for the development of many front-line drugs, yet our understanding of the relationship between attached sugars and biological activity is limited by the availability of convenient glycosylation methods. When a universal chemical glycosylation method that employs reducing sugars and requires no protection or activation is used, the glycorandomization of digitoxin leads to analogs that display significantly enhanced potency and tumor specificity and suggests a divergent mechanistic relationship between cardiac glycoside-induced cytotoxicity and Na+/K+-ATPase inhibition. This report highlights the remarkable advantages of glycorandomization as a powerful tool in glycobiology and drug discovery.

Animals↗
PubMed2005

Diversifying vancomycin via chemoenzymatic strategies.

[reaction: see text] 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.

Combinatorial Chemistry Techniques↗
PubMed2005

Structure-based engineering of E. coli galactokinase as a first step toward in vivo glycorandomization.

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.

Binding Sites↗
PubMed2005

Neoglycorandomization and chemoenzymatic glycorandomization: two complementary tools for natural product diversification.

In an effort to explore the contribution of the sugar constituents of pharmaceutically relevant glycosylated natural products, chemists have developed glycosylation methods that are amenable to the generation of libraries of analogues with a broad array of glycosidic attachments. Recently, two complementary glycorandomization strategies have been described, namely, neoglycorandomization, a chemical approach based on a one-step sugar ligation reaction that does not require any prior sugar protection or activation, and chemoenzymatic glycorandomization, a biocatalytic approach that relies on the substrate promiscuity of enzymes to activate and attach sugars to natural products. Since both methods require reducing sugars, this review first highlights recent advances in monosaccharide generation and then follows with an overview of recent progress in the development of neoglycorandomization and chemoenzymatic glycorandomization.

Aldehyde-Lyases↗