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Click-Chemistry-Based Free Azide versus Azido Sugar Detection Enables Rapid In Vivo Screening of Glycosynthase Activity
Glycosynthases are mutant glycosyl hydrolases that can synthesize glycosidic bonds between acceptor glycone/aglycone groups and activated donor sugars with suitable leaving groups (e.g., azido, fluoro). However, it has been challenging to rapidly detect glycosynthase reaction products involving azido sugars as donor sugars. This has limited our ability to apply rational engineering and directed evolution methods to rapidly screen for improved glycosynthases that are capable of synthesizing bespoke glycans. Here, we outline our recently developed screening methodologies for rapidly detecting glycosynthase activity using a model fucosynthase enzyme engineered to be active on fucosyl azide as donor sugar. We created a diverse library of fucosynthase mutants using semi-random and random error prone mutagenesis and then identified improved fucosynthase mutants with desired activity using two distinct screening methods developed by our group to detect glycosynthase activity (i.e., by detecting azide formed upon completion of fucosynthase reaction); (a) pCyn-GFP regulon method, and (b) Click chemistry method. Finally, we provide some proof-of-concept results illustrating the utility of both these screening methods to rapidly detect products of glycosynthase reactions involving azido sugars as donor groups.
Molecular docking is a computational method used to predict the preferred binding orientation of one molecule to another when bound to each other to form an energetically stable complex. This approach has been widely used for early-stage small-molecule drug design as well as identifying suitable protein-based macromolecule residues for mutagenesis. Estimating binding free energy, based on docking interactions of protein to its ligand based on an appropriate scoring function is often critical for protein mutagenesis studies to improve the activity or alter the specificity of targeted enzymes. However, calculating docking free energy for a large number of protein mutants is computationally challenging and time-consuming. Here, we showcase an end-to-end computational workflow for predicting the binding energy of pNP-Xylose substrate docked within the substrate binding site for a large library of combinatorial mutants of an alpha-L-fucosidase (TmAfc, PDB ID- 2ZWY) belonging to Thermotoga maritima glycosyl hydrolase (GH) family 29. Briefly, in silico combinatorial mutagenesis was performed for the top conserved residues in TmAfc as determined by running multiple sequence alignment against all GH29 family enzyme sequences downloaded from an in-house developed Carbohydrate-Active enZyme (CAZy) database retriever program. The binding energy was calculated through Autodock Vina with pNP-Xylose ligand docking with energy minimized TmAfc mutants, and the data was then used to train a neural network model which was also validated for model predictions using data from Autodock Vina. The current workflow can be adopted for any family of CAZymes to rapidly identify the effect of different mutations within the active site on substrate binding free energy to identify suitable targets for mutagenesis. We anticipate that this workflow could also serve as the starting point for performing more sophisticated and computationally intensive binding free energy calculations to identify targets for mutagenesis and hence optimize use of wet lab resources.
Data science and machine learning are revolutionizing enzyme engineering; however, high-throughput simulations for screening large libraries of enzyme variants remain a challenge. Here, we present a novel but highly simple approach to comparing enzyme variants with fully atomistic classical molecular dynamics (MD) simulations on a tractable timescale. Our method greatly simplifies the problem by restricting sampling only to the reaction transition state, and we show that the resulting measurements of transition-state stability are well correlated with experimental activity measurements across two highly distinct enzymes, even for mutations with effects too small to resolve with free energy methods. This method will enable atomistic simulations to achieve sampling coverage for enzyme variant prescreening and machine learning model training on a scale that was previously not possible.
To elucidate the relationship between carbohydrate structure and its biological function, glycoenzyme tools are widely applied in a variety of physiological and pathological scenarios. Intricate adjustment via glycoenzyme engineering improved the catalytic efficiency, extended the repertoire of accessible catalytic reactions, and tailored novel substrate specificities. In this review, we introduce the principles of glycoenzyme engineering and summarize the recent advances in the rational design and directed evolution of these enzymes, with an emphasize on screening methods in directed evolution. We also excerpt novel detection methods in glycobiology that can be adapted for the development of next-generation glycoenzyme tools.
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Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (>10 ⁷ mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the α1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency ( kcat / Km ) for the synthesis of Lewis x and 3′-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix α5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.
Combined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities. In this review, we intend to give a brief overview of the advantageous use of engineered glycoenzymes, sometimes in combination with chemical steps, for the synthesis of natural bioactive oligosaccharides or their precursors. The focus will be on examples resulting from the three main classes of glycoenzymes specialized in carbohydrate synthesis: glycosyltransferases, glycoside hydrolases and glycoside phosphorylases.
Glycosynthases are engineered mutant glycosidases that catalyse the formation of a glycosidic bond from a glycosyl donor and an acceptor alcohol. They are constructed by mutation of the enzymic nucleophile of a retaining glycosidase to a small non-nucleophilic residue. To date, five glycosynthases have been reported capable of synthesizing a range of β-glycosidic linkages. Methods to integrate protecting groups into glycosynthase-mediated glycosylations have been developed that broaden their applicability and enable finer control over product formation. Mutagenesis studies have improved the catalytic power of the original Abg glycosynthase, and a general methodology has been developed that allows the rapid screening of libraries of mutant glycosynthases for catalysts with improved activity. A method for determining aglycon substrate specificity has been developed to define the limits of substrate variation tolerated by a parent glycosidase and thence the derived glycosynthase. Together, these developments portend a bright future for the discovery of new glycosynthases and their widespread application as catalysts to assist in the rapid and efficient assembly of complex glycoconjugates.
The Carbohydrate-Active Enzymes database (CAZy; http://www.cazy.org) provides online and continuously updated access to a sequence-based family classification linking the sequence to the specificity
and 3D structure of the enzymes that assemble, modify and breakdown oligo- and polysaccharides. Functional and 3D structural
information is added and curated on a regular basis based on the available literature. In addition to the use of the database
by enzymologists seeking curated information on CAZymes, the dissemination of a stable nomenclature for these enzymes is probably
a major contribution of CAZy. The past few years have seen the expansion of the CAZy classification scheme to new families,
the development of subfamilies in several families and the power of CAZy for the analysis of genomes and metagenomes. This
article outlines the changes that have occurred in CAZy during the past 5 years and presents our novel effort to display the
resolution and the carbohydrate ligands in crystallographic complexes of CAZymes.
Here we present a new approach to aqueous hydrazoic acid detection through the synthesis and evaluation of an alkyne-based fluorescent probe, which could be applied in the monitoring of hydrazoic acid in both living Hela cells and larval zebrafish. This probe can also serve as an early warning automaton which would alert when both azide and protons exist over the threshold value.
Escherichia coli J53 (F− met pro Azir) is a derivative of E. coli K-12 which is resistant to sodium azide. This strain has been widely used as a general recipient strain for various conjugation
experiments. Here, we report the genome sequence of E. coli J53 (=KACC 16628).
An indole based naphthalene derivative is reported as a highly selective fluorescent probe for azide ion in aqueous ethanol. The probe is applied for cell imaging of the N(3)(-) ion in contaminated living cells. Both experimental and theoretical studies have been performed to figure out the plausible mechanism of fluorescence enhancement of the probe upon binding with N(3)(-).
Glycans are ubiquitous components of all organisms. Efforts to elucidate glycan function and to understand how they are assembled and disassembled can reap benefits in fields ranging from bioenergy to human medicine. Significant advances in our knowledge of glycan biosynthesis and function are emerging, and chemical biology approaches are accelerating the pace of discovery. Novel strategies for assembling oligosaccharides, glycoproteins, and other glycoconjugates are providing access to critical materials for interrogating glycan function. Chemoselective reactions that facilitate the synthesis of glycan-substituted imaging agents, arrays, and materials are yielding compounds to interrogate and perturb glycan function and dysfunction. To complement these advances, small molecules are being generated that inhibit key glycan-binding proteins or biosynthetic enzymes. These examples illustrate how chemical glycobiology is providing new insight into the functional roles of glycans and new opportunities to interfere with or exploit these roles.
Introducing structural modifications into biomolecules represents a powerful approach to dissect their functions and roles in biological processes. Bacterial polysaccharides, despite their rich structural information and essential roles in bacterium-host interactions and bacterial virulence, have largely been unexplored for in vivo structural modifications. In this study, we demonstrate the incorporation of a panel of monosaccharide analogs into bacterial polysaccharides in a highly homogenous manner via metabolic engineering of a promiscuous sugar nucleotide biosynthetic pathway. In addition, the bioorthorgonal functional groups metabolically incorporated were exploited for cell surface labeling using in vitro selective chemical ligation reactions. In summary, our study presents a general, facile and effective approach for in vivo generation of novel tailor-made bacterial polysaccharides.
The Carbohydrate-Active Enzyme (CAZy) database is a knowledge-based resource specialized in the enzymes that build and breakdown
complex carbohydrates and glycoconjugates. As of September 2008, the database describes the present knowledge on 113 glycoside
hydrolase, 91 glycosyltransferase, 19 polysaccharide lyase, 15 carbohydrate esterase and 52 carbohydrate-binding module families.
These families are created based on experimentally characterized proteins and are populated by sequences from public databases
with significant similarity. Protein biochemical information is continuously curated based on the available literature and
structural information. Over 6400 proteins have assigned EC numbers and 700 proteins have a PDB structure. The classification
(i) reflects the structural features of these enzymes better than their sole substrate specificity, (ii) helps to reveal the
evolutionary relationships between these enzymes and (iii) provides a convenient framework to understand mechanistic properties.
This resource has been available for over 10 years to the scientific community, contributing to information dissemination
and providing a transversal nomenclature to glycobiologists. More recently, this resource has been used to improve the quality
of functional predictions of a number genome projects by providing expert annotation. The CAZy resource resides at URL: http://www.cazy.org/.
Escherichia coli azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E. coli mutants isolated. Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E. coli plasma membrane. We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition. Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition. These studies point to the utility of specific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.
Fucosylated glycoconjugates are involved in numerous biological events, and alpha-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in alpha-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into alpha-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima alpha-l-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (beta/alpha)(8)-like domain and a C-terminal beta-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp(244), and the Brønsted acid/base, Glu(266). Because T. maritima alpha-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis.
Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ∼2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.
Techniques for synthesis of bespoke oligosaccharides currently lag behind those for other biopolymers such as polypeptides and polynucleotides, in part because of the paucity of satisfactory enzymatic tools to perform the synthetic reactions. One promising avenue of development for this problem is glycoside hydrolase enzymes with mutated nucleophile residues (called glycosynthases), which retain some elements of their native specificity and work with cheaply available substrates. However, the mechanistic underpinnings of this class of enzymes are not yet well-understood, and what few atomistic studies have been conducted have found different reaction pathways. In this paper, we describe the first unbiased computational study of the mechanism of a GH29 glycosynthase enzyme, Thermotoga maritima α-L-fucosidase (TmAfc) D224G. We find a single-step endothermic reaction step with an oxocarbenium-like transition state, demonstrating how stabilization of this transition state structure (which is common to many retaining glycoside hydrolases) can be repurposed in mutant enzymes to perform synthesis instead of hydrolysis. Our results are consistent with previous experimental observations and help both to clarify the mechanism of the existing single-mutant and to provide directions for further engineering of this and other glycosynthases.
Glycosylation is one of the most prevalent posttranslational modifications that profoundly affects the structure and functions of proteins in a wide variety of biological recognition events. However, the structural complexity and heterogeneity of glycoproteins, usually resulting from the variations of glycan components and/or the sites of glycosylation, often complicates detailed structure–function relationship studies and hampers the therapeutic applications of glycoproteins. To address these challenges, various chemical and biological strategies have been developed for producing glycan-defined homogeneous glycoproteins. This review highlights recent advances in the development of chemoenzymatic methods for synthesizing homogeneous glycoproteins, including the generation of various glycosynthases for synthetic purposes, endoglycosidase-catalyzed glycoprotein synthesis and glycan remodeling, and direct enzymatic glycosylation of polypeptides and proteins. The scope, limitation, and future directions of each method are discussed.
The structural and optical properties of hydroxyphenyl-substituted-1,2,3-triazole molecules (“click” triazoles) are described. “Click” triazoles are prepared from the copper(I)-catalyzed azide-alkyne cycloaddition reactions. The alkyne-derived C4 substituent of a “click” triazole engages in electronic conjugation more effectively with the triazolyl core than the azide-derived N1 substituent. Furthermore, triazolyl group exerts a stronger electron-withdrawing effect on the N1 than the C4 substituent. Therefore, the placement of an electron-donating group at either C4 or N1 position, and the presence or the absence of an intramolecular hydrogen bond (HB), have profound influences on the optical properties of these compounds. The reported “click” triazoles have fluorescence quantum yields in the range of 0.1-0.3 and large apparent Stokes shifts (8,000-13,000 cm-1) in all tested solvents. Deprotonation of “click” triazoles with a C4 hydroxyphenyl group increases their Stokes shifts; while the opposite (or quenching) occurs to the triazoles with an N1 hydroxyphenyl substituent. For the triazoles that contain intramolecular HBs, neither experimental nor computational results support a model of excited state intramolecular proton transfer (ESIPT). Rather, the excited state internal (or intramolecular) charge transfer (ICT) mechanism is more suitable to explain the fluorescence properties of the hydroxyphenyl-substituted “click” triazoles; specifically, the large Stokes shifts of these compounds.
Little is known about the reactivity of strain-promoted alkyne-azide cycloaddition (SPAAC) reagents with inorganic azides. We explore the reactions of a variety of popular SPAAC reagents with sodium azide and hydrozoic acid. We find that the reactions proceed in water at rates comparable to those with organic azides, yielding in all cases a triazole adduct. The azide ion's utility as a cyclooctyne quenching reagent is demonstrated by using it to spatially pattern uniformly-doped hydrogels. The facile quenching of cyclooctynes demonstrated here should be useful in other bioorthogonal ligation techniques in which cyclooctynes are employed, including SPANC, Diels-Alder, and thiol-yne.
The synthesis of glycosidic structures by catalysis via glycosynthases has gained much interest due to the potential high product yields and specificity of the enzymes. Nevertheless, the characterisation and implementation of new glycosynthases is greatly hampered by the lack of high-throughput methods for reaction analysis and screening of potential glycosynthase variants. Fluoride detection, via silyl ether chemosensors, has recently shown high potential for the identification of glycosynthase mutants in a high-throughput manner, though limited by the low maximal detection concentration. In the present paper, we describe a new version of a glycosynthase activity assay using a silyl ether of p-nitrophenol, allowing fast reliable detection of fluoride even at concentrations of 4mm and higher. This improvement of detection allows not only screening and identification but also kinetic characterisation of glycosynthases and synthetic reactions in a fast microtiter plate format. The applicability of the assay was successfully demonstrated by the biochemical characterisation of the mesophilic β-glucosynthase of Abg-E358S (Rhizobium radiobacter) and psychrotolerant β-glucosynthase BglU-E377A (Micrococcus antarcticus). The limitation of hyperthermophilic glycosidases as potential glycosynthases, when using glycosyl fluoride donors, was also illustrated by the example of the putative β-galactosidase GalPf from Pyrococcus furiosus.
To shed light on intramolecular charge-transfer phenomena in 1,2,3-triazole-linked materials, a series of 1,2,3-triazole-linked push-pull chromophores were prepared and studied experimentally and computationally. Investigated modifications include variation of donor and/or acceptor strength and linker moiety as well as regioisomers. Photophysical characterization of intramolecular charge-transfer features revealed ambipolar behavior of the triazole linker, depending on the substitution position. Furthermore, non-centrosymmetric materials were subjected to second-harmonic generation measurements, which revealed the high nonlinear optical activity of this class of materials.
Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC), popularly known as the "click reaction", serves as the most potent and highly dependable tool for facile construction of simple to complex architectures at the molecular level. Click-knitted threads of two exclusively different molecular entities have created some really interesting structures for more than 15 years with a broad spectrum of applicability, including in the fascinating fields of synthetic chemistry, medicinal science, biochemistry, pharmacology, material science, and catalysis. The unique properties of the carbohydrate moiety and the advantages of highly chemo- and regioselective click chemistry, such as mild reaction conditions, efficient performance with a wide range of solvents, and compatibility with different functionalities, together produce miraculous neoglycoconjugates and neoglycopolymers with various synthetic, biological, and pharmaceutical applications. In this review we highlight the successful advancement of Cu(I)-catalyzed click chemistry in glycoscience and its applications as well as future scope in different streams of applied sciences.
Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is
growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-l-Fucosidases catalyze the hydrolysis of α-l-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family
fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated
that the α-l-fucosidase from Thermotoga maritima (TmαFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution (
Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-l-transfucosidase starting with the α-l-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a
strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation
reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated
HMOs.
The diversity of oligo- and polysaccharides provides an abundance of biological roles for these carbohydrates. The enzymes hydrolysing these compounds, the glycoside hydrolases, therefore mediate a wealth of biological functions. Glycoside hydrolases fall into a number of sequence-based families. The recent analysis of these families, coupled with the burgeoning number of 3D structures, provides a detailed insight into the structure, function and catalytic mechanism of these enzymes.
Computing in science and engineering is now ubiquitous: digital technologies underpin, accelerate, and enable new, even transformational, research in all domains. Access to an array of integrated and well-supported high-end digital services is critical for the advancement of knowledge. Driven by community needs, the Extreme Science and Engineering Discovery Environment (XSEDE) project substantially enhances the productivity of a growing community of scholars, researchers, and engineers (collectively referred to as "scientists"' throughout this article) through access to advanced digital services that support open research. XSEDE's integrated, comprehensive suite of advanced digital services federates with other high-end facilities and with campus-based resources, serving as the foundation for a national e-science infrastructure ecosystem. XSEDE's e-science infrastructure has tremendous potential for enabling new advancements in research and education. XSEDE's vision is a world of digitally enabled scholars, researchers, and engineers participating in multidisciplinary collaborations to tackle society's grand challenges.
Streptomyces sp. SirexAA-E (ActE) has been identified as a highly cellulolytic actinobacterium capable of deconstructing lignocellulosic biomass. SirexAA-E CAZymes most frequently contain a carbohydrate-binding module from family 2a (CBM2a). The DNA encoding the CBM2a from gene locus SACTE_0237, the most abundantly expressed cellulase from SirexAA-E, was cloned into an Escherichia coli expression vector and expressed as a C-terminal fusion protein to GFP. The GFP-CBM2a fusion protein was purified from insoluble inclusion bodies and refolded. The solubilized protein was separated by size-exclusion chromatography into high molecular weight GFP-CBM2a multimers and monomeric GFP-CBM2a. Only the monomeric CBM2a protein was found to have high relative affinity (partition coefficient of 0.62ｱ0.04 liters/gram) to cellulose. Binding of monomeric CBM2a prepared in this manner exhibits fully reversible, high affinity binding to cellulose.
Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. We here report a general screening assay independent of the enzyme specificity, and implemented in HTS format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the Glu (catalytic) nucleophile, other variants have shown to acquire glycosynthases activity. Surprisingly, an Asp for Glu replacement renders a highly active glycosynthases, but still retaining low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase.
Chemical glycobiology is emerging as one of the most uniquely powerful sub-disciplines of chemical biology. The previous scarcity of chemical strategies and the unparalleled structural diversity have created a uniquely fertile ground that is both rich in challenges and potentially very profound in implications. Glycans (oligosaccharides, polysaccharides, and glycoconjugates) are everywhere in biological systems and yet remain disproportionately neglected - reviews highlighting this 'Cinderella status' abound. Yet, the last two decades have witnessed tremendous progress, notably in chemical and chemoenzymatic synthesis, 'sequencing' and arraying, metabolic engineering and imaging. These vital steps serve to highlight not only the great potential but just how much more remains to be done. The vast chemical and functional space of glycans remains to be truly explored. Top-down full-scale glycomic and glycoproteomic studies coupled with hypothesis-driven, bottom-up innovative chemical strategies will be required to properly realize the potential impact of glycoscience on human health, energy, and economy. In this review, we cherry-pick far-sighted advances and use these to identify possible challenges, opportunities and avenues in chemical glycobiology.
The beta-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus hydrolyzes beta-glycosides following a retaining mechanism based upon the action of two amino acids: Glu387, which acts as the nucleophile of the reaction, and Glu206, which acts as the general acid/base catalyst. The activities of inactive mutants of the catalytic nucleophile Glu387Ala/Gly were restored by externally added nucleophiles. Sodium azide and sodium formate were used as external nucleophiles and the products of their reaction were characterized. Glu387Ala/Gly mutants were reactivated with 2, 4-DNP-beta-Glc substrate and the Glu387Gly mutant showed recovered activity, with the same nucleophiles, also on 2-NP-beta-Glc. The reaction catalyzed by the Glu387Gly mutant proceeded differently depending on the type of externally added nucleophile. Sodium azide restored the catalytic activity of the mutant by attacking the alpha-side of the anomeric carbon of the substrates, thereby yielding an inverting glycosidase. Sodium formate promoted the opposite behavior (retaining) in the mutant, producing 3-O-beta-linked disaccharide derivative of the substrates. A possible role of sodium formate as a biomimicking agent in replacing the natural nucleophile Glu387 is also discussed.
Background: Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered. The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly. An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme. A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes.Results: Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar. A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants.Conclusions: Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases.
Removal of the catalytic nucleophile Glu134 of the retaining 1,3-1,4-β-glucanase from Bacillus licheniformis by mutation to alanine yields an enzyme with no glycosidase activity. The mutant is able to catalyze the regio- and stereospecific glycosylation of α-laminaribiosyl fluoride with different glucoside acceptors through a single-step inverting mechanism. The main advantage of the mutant as glycosylation catalyst with respect to the kinetically controlled transglycosylation using the wild-type enzyme is that the reaction products cannot be hydrolyzed by the mutant enzyme, and glycosylation yields rise to 90%.
Iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of amylosucrase from Neisseria polysaccharea, a sucrose-utilizing α-transglucosidase, and sort out improved variants. From the screening of three semi-rational sub-libraries accounting in total for 20,000 variants, we report here the isolation of three double-mutants of N. polysaccharea amylosucrase displaying a spectacular specificity enhancement towards both sucrose, the donor substrate, and the allyl 2-acetamido-2-deoxy-α-D-glucopyranoside acceptor compared to the wild-type enzyme. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structure of the best performing enzymes supported by molecular dynamics simulations showed local rigidity of the -1 subsite as well as flexibility of loops involved in active site topology, which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process, when designing enzyme libraries.
Glycosynthases, engineered glycoside hy-drolases that are able to synthesize glycans in quanti-tative yields without hydrolyzing them, are among the most interesting tools for the chemoenzymatic synthesis of carbohydrates that have been made available so far. From their invention in 1998, these enzymes have been developed enormously and dem-onstrated to be convenient alternatives to the more expensive glycosyltransferase. Glycosynthases have been proved to be efficient catalysts for the synthesis of oligosaccharides, polysaccharides, and glycoconju-gates of various lengths containing different mono-saccharides bound through a-and b-linkages, which have interesting applications. In this review we de-scribe the impact that glycosynthases have had on the biocatalysis and biosynthesis of carbohydrates through the description of the novel activities pro-duced and of the development of the glycosynthase-based process. In addition, we summarize the ap-proaches that, according to the current literature, can be adopted to produce novel glycosynthase activ-ities and to improve the existing process.
Oligo-, polysaccharides, and glycoconjugates are a relevant part of the bioactive components of the natural products exploited in therapeutics, diagnostics, food additives, and biomaterials. Glycans are directly involved in important biological processes, such as immunostimulation, anti-inflammatory, antioxidant, and chemoprotectant actions and/or are crucial for their activity, by modulating target recognition, stability, and pharmacokinetics. On the other hand, carbohydrate extracts used for functional studies are rather heterogeneous and lack structural information because of their intrinsic complexity hampering purification and characterization. Therefore, methods for glycoside synthesis and modification are urgently needed. Recently, glycosynthases, engineered glycoside hydrolases with no hydrolytic activity that synthesize glycans in quantitative yields, were introduced. Here we will illustrate how the glycosynthases described so far might be exploited for the production of glycan analogs of natural products and their enormous potential in this field.
Protein glycosylation is a common and complex posttranslational modification of proteins, which expands functional diversity while boosting structural heterogeneity. Glycoproteins, the end products of such a modification, are typically produced as mixtures of glycoforms possessing the same polypeptide backbone but differing in the site of glycosylation and/or in the structures of pendant glycans, from which single glycoforms are difficult to isolate. The urgent need for glycan-defined glycoproteins in both detailed structure-function relationship studies and therapeutic applications has stimulated an extensive interest in developing various methods for manipulating protein glycosylation. This review highlights emerging technologies that hold great promise in making a variety of glycan-defined glycoproteins, with a particular emphasis in the following three areas: specific glycoengineering of host biosynthetic pathways, in vitro chemoenzymatic glycosylation remodeling, and chemoselective and site-specific glycosylation of proteins.
Understanding how enzymes mediate catalysis is a key to their reprogramming for biotechnological applications. The family 3 retaining glycosidase postulated to be involved in erythromycin self-resistance was cloned, recombinantly expressed in Escherichia coli, purified, and characterized. Bioinformatics analysis allowed the identification of the acid/base and nucleophile residues, and mutation of these residues resulted in hydrolytically inactive proteins. One mutant was able to synthesize a glycosidic linkage using α-glucosyl fluoride as a donor and macrolide antibiotics as acceptors. This shows an unprecedented application of glycosynthase technology in accomplishing a challenging β-(1,2)-glycosylation of an amino sugar. This work also provides the first biochemical characterization of the EryBI protein and supports its role in the self-resistance mechanism involved in erythromycin biosynthesis. An in vivo selection approach was used in an attempt to spur evolution of the glycosynthase, and the results from the attempted selection method provide insight into the requirements for in vivo directed evolution of glycosynthases.
Bioorthogonal chemistry allows a wide variety of biomolecules to be specifically labeled and probed in living cells and whole organisms. Here we discuss the history of bioorthogonal reactions and some of the most interesting and important advances in the field.
Glycosyltransferases catalyze glycosidic bond formation using sugar donors containing a nucleoside phosphate or a lipid phosphate leaving group. Only two structural folds, GT-A and GT-B, have been identified for the nucleotide sugar-dependent enzymes, but other folds are now appearing for the soluble domains of lipid phosphosugar-dependent glycosyl transferases. Structural and kinetic studies have provided new insights. Inverting glycosyltransferases utilize a direct displacement S(N)2-like mechanism involving an enzymatic base catalyst. Leaving group departure in GT-A fold enzymes is typically facilitated via a coordinated divalent cation, whereas GT-B fold enzymes instead use positively charged side chains and/or hydroxyls and helix dipoles. The mechanism of retaining glycosyltransferases is less clear. The expected two-step double-displacement mechanism is rendered less likely by the lack of conserved architecture in the region where a catalytic nucleophile would be expected. A mechanism involving a short-lived oxocarbenium ion intermediate now seems the most likely, with the leaving phosphate serving as the base.
Bioconjugation techniques using organic azides are compared in this critical review. A particular focus is on chemical ligation reactions and their application to chemical biology (179 references).
The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.
The expanding field of glycobiology requires tools for the synthesis of structurally defined oligosaccharides and glycoconjugates, while any potential therapeutic applications of sugar-based derivates would require access to substantial quantities of such compounds. Classical chemical approaches are not well suited for such large-scale syntheses, thus enzymatic approaches are sought. Traditional routes to the enzymatic assembly of oligosaccharides have involved the use of either Nature's own biosynthetic enzymes, the glycosyl transferases, or glycosidases run in transglycosylation mode. However, each approach has drawbacks that have limited its application. Glycosynthases are mutant glycosidases in which the catalytic nucleophile has been replaced by mutation, inactivating them as hydrolases. When used in conjunction with glycosyl fluorides of the opposite anomeric configuration to that of the substrate, these enzymes function as highly efficient transferases, frequently giving stoichiometric yields of products. Further improvements can be obtained through directed evolution of the gene encoding the enzyme in question, but this requires the ability to screen very large libraries of catalysts. In this review we survey new screening methods for the formation of glycosidic linkages using high-throughput techniques, such as FACS, chemical complementation, and robot-assisted ELISA assays. Enzymes were evolved to have higher catalytic activity with their natural substrates, to show altered substrate specificities or to be promiscuous for efficient application in oligosaccharide, glycolipid, and glycoprotein synthesis.
Synthetic oligosaccharides and glycoconjugates are increasingly used as probes for biological research and as lead compounds for drug and vaccine discovery. These endeavors are, however, complicated by a lack of general methods for the routine preparation of this important class of compounds. Recent development such as one-pot multi-step protecting group manipulations, the use of unified monosaccharide building blocks, the introduction of stereoselective glycosylation protocols, and convergent strategies for oligosaccharide assembly, are beginning to address these problems. Furthermore, oligosaccharide synthesis can be facilitated by chemo-enzymatic methods, which employ a range of glycosyl transferases to modify a synthetic oligosaccharide precursor. Glycosynthases, which are mutant glycosidases, that can readily form glycosidic linkages are addressing a lack of a wide range glycosyltransferases. The power of carbohydrate chemistry is highlighted by an ability to synthesize glycoproteins.
Fucose-containing oligosaccharides play a central role in physio-pathological events, and fucosylated oligosaccharides have interesting potential applications in biomedicine. No methods for the large-scale production of oligosaccharides are currently available, but the chemo-enzymatic approach is very promising. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, have been demonstrated to be an interesting alternative. However, examples of glycosynthases available so far are restricted to a limited number of glycosidases families and to only one retaining alpha-glycosynthase. We show here that new mutants of two alpha-L-fucosidases are efficient alpha-L-fucosynthases. The approach shown utilized beta-L-fucopyranosyl azide as donor substrate leading to transglycosylation yields up to 91%. This is the first method exploiting a beta-glycosyl azide donor for alpha-glycosynthases; its applicability to the glycosynthetic methodology in a wider perspective is presented.
Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immune diseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in their synthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screening strategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, and we identified several mutants with enhanced activities. These new catalysts, which are capable of producing a broad range of homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids and demonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.
Various beta-glycosyl azides have been synthesized directly in water by the reaction of unprotected sugars and sodium azide mediated by 2-chloro-1,3-dimethylimidazolinium chloride (DMC).
Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-beta-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20 g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms.
Directed evolution can generate a remarkable range of new enzyme properties. Alternate substrate specificities and reaction selectivities are readily accessible in enzymes from families that are naturally functionally diverse. Activities on new substrates can be obtained by improving variants with broadened specificities or by step-wise evolution through a sequence of more and more challenging substrates. Evolution of highly specific enzymes has been demonstrated, even with positive selection alone. It is apparent that many solutions exist for any given problem, and there are often many paths that lead uphill, one step at a time.
The diversity of oligo- and polysaccharides provides an abundance of biological roles for these carbohydrates. The enzymes hydrolysing these compounds, the glycoside hydrolases, therefore mediate a wealth of biological functions. Glycoside hydrolases fall into a number of sequence-based families. The recent analysis of these families, coupled with the burgeoning number of 3D structures, provides a detailed insight into the structure, function and catalytic mechanism of these enzymes.
Traditionally, access to structurally defined complex carbohydrates has been very laborious. Although recent advancements
in solid-phase synthesis have made the construction of complex oligosaccharides less tedious, a high level of technical expertise
is still necessary to obtain the desired structures. We describe the automated chemical synthesis of several oligosaccharides
on a solid-phase synthesizer. A branched dodecasaccharide was synthesized through the use of glycosyl phosphate building blocks
and an octenediol functionalized resin. The target oligosaccharide was readily obtained after cleavage from the solid support.
Access to certain complex oligosaccharides now has become feasible in a fashion much like the construction of oligopeptides
and oligonucleotides.
The Streptomyces sp. beta-glucosidase (Bgl3) is a retaining glycosidase that belongs to family 1 glycosyl hydrolases. Steady-state kinetics with p-nitrophenyl beta-D-glycosides revealed that the highest k(cat)/K(M) values are obtained with glucoside (with strong substrate inhibition) and fucoside (with no substrate inhibition) substrates and that Bgl3 has 10-fold glucosidase over galactosidase activity. Reactivity studies by means of a Hammett analysis using a series of substituted aryl beta-glucosides gave a biphasic plot log k(cat) vs pK(a) of the phenol aglycon: a linear region with a slope of beta(lg) = -0.8 for the less reactive substrates (pK(a) > 8) and no significant dependence for activated substrates (pK(a) < 8). Thus, according to the two-step mechanism of retaining glycosidases, formation of the glycosyl-enzyme intermediate is rate limiting for the former substrates, while hydrolysis of the intermediate is for the latter. To identify key catalytic residues and on the basis of sequence similarity to other family 1 beta-glucosidases, glutamic acids 178 and 383 were changed to glutamine and alanine by site-directed mutagenesis. Mutation of Glu178 to Gln and Ala yielded enzymes with 250- and 3500-fold reduction in their catalytic efficiencies, whereas larger reduction (10(5)-10(6)-fold) were obtained for mutants at Glu383. The functional role of both residues was probed by a chemical rescue methodology based on activation of the inactive Ala mutants by azide as exogenous nucleophile. The E178A mutant yielded the beta-glucosyl azide adduct (by (1)H NMR) with a 200-fold increase on k(cat) for the 2,4-dinitrophenyl glucoside but constant k(cat)/K(M) on azide concentration. On the other hand, the E383A mutant with the same substrate gave the alpha-glucosyl azide product and a 100-fold increase in k(cat) at 1 M azide. In conclusion, Glu178 is the general acid/base catalyst and Glu383 the catalytic nucleophile. The results presented here indicate that Bgl3 beta-glucosidase displays kinetic and mechanistic properties similar to other family 1 enzymes analyzed so far. Subtle differences in behavior would lie in the fine and specific architecture of their respective active sites.
Recently, we reported a general assay for enzyme catalysis based on the yeast three-hybrid assay, Chemical Complementation, which is intended to expand the range of chemical reactions to which directed evolution can be applied. Here, Chemical Complementation was applied to a glycosynthase derived from a retaining glycosidase, an important class of enzymes for carbohydrate synthesis. Using the yeast three-hybrid assay, the glycosynthase activity of the E197A mutant of the Cel7B from Humicola insolens was linked to transcription of a LEU2 reporter gene, making cell growth dependent on glycosynthase activity in the absence of leucine. Then the LEU2 selection was used to isolate the most active glycosynthase from a Glu197 saturation library, yielding an E197S Cel7B variant with a 5-fold increase in glycosynthase activity. These results not only establish Chemical Complementation as a platform for the directed evolution of glycosynthases, but also show the generality of this approach and the ease with which it can be applied to new chemical reactions.