232 research outputs found
Trapping and Characterization of the Reaction Intermediate in Cyclodextrin Glycosyltransferase by Use of Activated Substrates and a Mutant Enzyme
Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration. They are also shown to catalyze the coupling of maltooligosaccharyl fluorides. Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. This intermediate can be trapped by use of 4-deoxymaltotriosyl α-fluoride (4DG3αF). This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing. When 4DG3αF was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (kcat = 2 s-1) compared with the parent glycosyl fluoride, maltotriosyl R-fluoride (kcat = 275 s-1). Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification. However, when 4DG3αF was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (kcat = 0.6 s-1). Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry. Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase. Asp229 is found to be conserved in all members of the family 13 glycosyl transferases.
Functional metagenomic screening approach for discovery of new glycoside phosphorylases
Glycoside phosphorylases (GPs) have recently been recognized as potentially useful biocatalysts for the synthesis and biotransformation of glycans. These enzymes ordinarily carry out phosphorolysis of the glycosidic linkage by transferring a glycosyl moiety from the non-reducing end of a di- or polysaccharide substrate onto inorganic phosphate, thereby cleaving the glycosidic bond and generating a sugar-1-phosphate. GPs distinguish themselves from most carbohydrate-active enzymes in that the hydrolytic free energy associated with the ester-linkage of the sugar-1-phosphate product is roughly equivalent to that of the glycosidic linkage in the glycan substrate. Therefore, the equilibrium position can be tipped in favour of glycoside synthesis by manipulation of reaction conditions. GPs thus have considerable potential for the assembly of glycans, especially since their reversibility would allow the use of one GP to degrade an inexpensive glycan to produce a pool of sugar-1-phosphates, while a second GP could be deployed to use those sugar-1-phosphates as donors to synthesize a different, more valuable target glycan. The bottleneck in this approach, however, is the limited range of GPs available, which restricts the classes of glycan that can be assembled. To help increase the spectrum of known GPs available, we have turned to metagenomics as a means to discover new enzymes belonging to this class. We have adapted the molybdenum blue reaction to a high-throughput plate-based metagenomic screen for the discovery of GPs. Our method utilizes the reverse phosphorolysis ability of GPs by coupling inorganic phosphate released during glycan synthesis to the molybdenum blue reaction. Therefore, GP activity can be identified by incubating metagenomic clones with appropriate donor sugar-1-phosphates and acceptor glycans, then monitoring inorganic phosphate accumulation by measuring formation of molybdenum blue. Our pilot screen was optimized to identify cellulose degrading GPs and yielded 7 novel GP ORFs, all from CAZy family GH94. To our knowledge this is the first high-throughput functional metagenomic screen for GP activity. Looking ahead, we have planned to further adapt the screening method so it may identify GP activity from other CAZy families, beyond GH94. The activities that can be identified are dependent on the combination of donor and acceptor substrates used. By mixing and matching different substrates we will be able to narrow or broaden the scope of activities that can be detected within a single screen
Reassessment of Acarbose as a Transition State Analogue Inhibitor of Cyclodextrin Glycosyltransferase
The binding of several different active site mutants of Bacillus circulans cyclodextrin glycosyltransferase to the inhibitor acarbose has been investigated through measurement of Ki values. The mutations represent several key amino acid positions, most of which are believed to play important roles in governing the product specificity of cyclodextrin glycosyltransferase. Michaelis-Menten parameters for the substrates α-maltotriosyl fluoride (αG3F) and α-glucosyl fluoride (αGF) with each mutant have been determined by following the enzyme-catalyzed release of fluoride with an ion-selective fluoride electrode. In both cases, reasonable correlations are observed in logarithmic plots relating the Ki value for acarbose with each mutant and both kcat/Km and Km for the hydrolysis of either substrate by the corresponding mutants. This indicates that acarbose, as an inhibitor, is mimicking aspects of both the ground state and the transition state. A better correlation is observed for αGF (r = 0.98) than αG3F (r = 0.90), which can be explained in terms of the modes of binding of these substrates and acarbose. Re-refinement of the previously determined crystal structure of wild-type CGTase complexed with acarbose reveals a binding mode consistent with the transition state analogue character of this inhibitor.
Directed evolution of glycosyltransferase for the artificial biosynthesis of natural product glycosides
Over one fifth natural product drugs (including protein biopharmaceuticals), cosmetics, and nutraceuticals have a diverse set of sugars in their structures. These glycosylations dramatically influence the physicochemical and pharmacological properties of these compounds. Glycosyltransferases (GTs) offer very attractive approaches to the biosynthesis of complex glycosylated natural products. However, the limited number of available GTs, together with their instability and strict substrate specificity, have severely hampered the broad application of these enzymes. In the past few years, we have used directed evolution as a tool to tailor the GTs with desired substrate specificity and higher catalytic activity. Here I will introduce some of our efforts in 1) the semi-rational design of a glucosyltransferase UGT51 from S. cerevisiae to repurposing its promiscuous activity towards the biosynthesis of rare ginsenoside Rh2; and 2) the directed evolution of an α1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method. I will also discuss the development of new tools for the high-throughput screening method for GTs and the mechanistic insight we found during the evolution of these enzymes
High-throughput substrate specificity analysis of metagenomic-derived arabinoxylan-active enzymes
High-Throughput Screening for Human Lysosomal β-N-Acetyl Hexosaminidase Inhibitors Acting as Pharmacological Chaperones
SummaryThe adult forms of Tay-Sachs and Sandhoff diseases result when the activity of β-hexosaminidase A (Hex) falls below ∼10% of normal due to decreased transport of the destabilized mutant enzyme to the lysosome. Carbohydrate-based competitive inhibitors of Hex act as pharmacological chaperones (PC) in patient cells, facilitating exit of the enzyme from the endoplasmic reticulum, thereby increasing the mutant Hex protein and activity levels in the lysosome 3- to 6-fold. To identify drug-like PC candidates, we developed a fluorescence-based real-time enzyme assay and screened the Maybridge library of 50,000 compounds for inhibitors of purified Hex. Three structurally distinct micromolar competitive inhibitors, a bisnaphthalimide, nitro-indan-1-one, and pyrrolo[3,4-d]pyridazin-1-one were identified that specifically increased lysosomal Hex protein and activity levels in patient fibroblasts. These results validate screening for inhibitory compounds as an approach to identifying PCs
High-throughput generation of product profiles for arabinoxylan-active enzymes from metagenomes
Metagenomics is an exciting alternative to seek carbohydrate-active enzymes from a range of sources. Typically, metagenomics reveals dozens of putative catalysts that require functional characterization for further application in industrial processes. High-throughput screening methods compatible with adequate natural substrates are crucial for an accurate functional elucidation of substrate preferences. Based on DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) analysis of enzymatic-reaction products, we generated product profiles to consequently infer substrate cleavage positions, resulting in the generation of enzymatic-degradation maps. Product profiles were produced in high throughput for arabinoxylan (AX)-active enzymes belonging to the glycoside hydrolase families GH43 (subfamilies 2 [MG432], 7 [MG437], and 28 [MG4328]) and GH8 (MG8) starting from 12 (arabino)xylo-oligosaccharides. These enzymes were discovered through functional metagenomic studies of feces from the North American beaver (Castor canadensis). This work shows how enzyme loading alters the product profiles of all enzymes studied and gives insight into AX degradation patterns, revealing sequential substrate preferences of AX-active enzymes.
IMPORTANCE: Arabinoxylan is mainly found in the hemicellulosic fractions of rice straw, corn cobs, and rice husk. Converting arabinoxylan into (arabino)xylooligosaccharides as added-value products that can be applied in food, feed, and cosmetics presents a sustainable and economic alternative for the biorefinery industries. Efficient and profitable AX degradation requires a set of enzymes with particular characteristics. Therefore, enzyme discovery and the study of substrate preferences are of utmost importance. Beavers, as consumers of woody biomass, are a promising source of a repertoire of enzymes able to deconstruct hemicelluloses into soluble oligosaccharides. High-throughput analysis of the oligosaccharide profiles produced by these enzymes will assist in the selection of the most appropriate enzymes for the biorefinery
A Novel Anti-diabetic Metabolite from Plants: Biosynthesis, Gene Discovery, and Metabolic Engineering of Montbretin A
Plant specialized metabolites (i.e. secondary metabolites) have been employed by humans for centuries in traditional and modern medicine. They remain an important source for the discovery of new pharmaceuticals and nutraceuticals. Montbretin A (MbA) is a complex acylated flavonoid glycoside discovered in the below-ground storage organs (corms) of the ornamental plant montbretia (Crocosmia x crocosmiiflora). MbA a highly potent and selective inhibitor of the human pancreatic α-amylase (HPA), a key enzyme in starch degradation. MbA is being tested for the treatment of type-2 diabetes. However, due to low abundance of MbA in montbretia plants and due the complex chemical structure of MbA, natural product extraction and chemical synthesis are insufficient for MbA production. Our goal is to develop a heterologous plant production system or a microbial production system for MbA. This requires knowledge of the genes, enzymes and regulating factors of the MbA biosynthetic system in montbretia. We achieved the discovery of the complete biosynthetic pathway of MbA using an approach that combined knowledge of montbretia biology, metabolite profiling, differential transcriptome analysis, cDNA cloning, heterologous gene expression in E. coli, yeast and tobacco, and enzyme biochemistry. This includes the discovery of five new UDP-sugar dependent glycosyltransferases (UGTs) and a BAHD-acyltransferases (AT) which together catalyze the complete assembly of MbA from its different building blocks. To reconstruct MbA production in tobacco (Nicotiana benthamiana) we enhanced the biosynthesis of flavonol precursors using genes for myricetin biosynthesis and transcription factors from montbtretia, which were stacked with genes of the MbA assembly pathway. We will highlight both challenges and opportunities of exploring novel biosynthetic systems of plant specialized metabolites for the development of new drugs, and bioproducts in general
Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue *
Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate
Performing statistical analyses on quantitative data in Taverna workflows: an example using R and maxdBrowse to identify differentially-expressed genes from microarray data.
BACKGROUND: There has been a dramatic increase in the amount of quantitative data derived from the measurement of changes at different levels of biological complexity during the post-genomic era. However, there are a number of issues associated with the use of computational tools employed for the analysis of such data. For example, computational tools such as R and MATLAB require prior knowledge of their programming languages in order to implement statistical analyses on data. Combining two or more tools in an analysis may also be problematic since data may have to be manually copied and pasted between separate user interfaces for each tool. Furthermore, this transfer of data may require a reconciliation step in order for there to be interoperability between computational tools. RESULTS: Developments in the Taverna workflow system have enabled pipelines to be constructed and enacted for generic and ad hoc analyses of quantitative data. Here, we present an example of such a workflow involving the statistical identification of differentially-expressed genes from microarray data followed by the annotation of their relationships to cellular processes. This workflow makes use of customised maxdBrowse web services, a system that allows Taverna to query and retrieve gene expression data from the maxdLoad2 microarray database. These data are then analysed by R to identify differentially-expressed genes using the Taverna RShell processor which has been developed for invoking this tool when it has been deployed as a service using the RServe library. In addition, the workflow uses Beanshell scripts to reconcile mismatches of data between services as well as to implement a form of user interaction for selecting subsets of microarray data for analysis as part of the workflow execution. A new plugin system in the Taverna software architecture is demonstrated by the use of renderers for displaying PDF files and CSV formatted data within the Taverna workbench. CONCLUSION: Taverna can be used by data analysis experts as a generic tool for composing ad hoc analyses of quantitative data by combining the use of scripts written in the R programming language with tools exposed as services in workflows. When these workflows are shared with colleagues and the wider scientific community, they provide an approach for other scientists wanting to use tools such as R without having to learn the corresponding programming language to analyse their own data.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are
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