Hong-Ming Chen

High-throughput screening methodology for the directed evolution of glycosyltransferases.

Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of new oligosaccharides holds great potential for the development of the field of glycobiology. However, engineering of GTs by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of a new fluorescence-based high-throughput screening (HTS) methodology for the directed evolution of sialyltransferases (STs). Using this methodology, we detected the formation of sialosides in intact Escherichia coli cells by selectively trapping the fluorescently labeled transfer products in the cell and analyzing and sorting the resulting cell population using a fluorescence-activated cell sorter (FACS). We screened a library of >106 ST mutants using this methodology and found a variant with up to 400-fold higher catalytic efficiency for transfer to a variety of fluorescently labeled acceptor sugars, including a thiosugar, yielding a metabolically stable product.

Mechanism-Based Covalent Neuraminidase Inhibitors with Broad Spectrum Influenza Antiviral Activity

Adding to the Antiviral Arsenal The envelope of influenza virus contains two immunodominant glycoproteins: hemagglutinin and neuraminidase (NA). Existing antivirals like zanamivir (Relenza) and oseltamivir (Tamiflu) target NA; however, the development of drug resistance is a problem. Kim et al. (p. 71, published online 21 February) now report a different class of NA inhibitors. NA catalyzes the removal of sialic acids from the surface of host cells to initiate entry. Discovery of a NA–sialic acid intermediate led to the production of sialic acid analogs that bound covalently to NA and inhibited its enzymatic activity. These compounds showed activity against a wide variety of influenza strains, inhibited viral replication in cell culture, and were able to protect mice against influenza infection. Protection of mice was equivalent to protection seen from zanamivir. Moreover, the compounds showed activity against drug-resistant strains in vitro. These compounds represent a potentially useful addition to the arsenal of antivirals used to treat influenza infection. Looking deeply into the mechanism of enzyme inhibition provides a clue for the development of new drugs to fight flu. Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.

Self‐Immobilizing Fluorogenic Imaging Agents of Enzyme Activity

Chromogenic and fluorogenic substrates are valuable tools for locating endogenousand reporter-enzyme activities, and thus for visualizing gene expression within cells and tissues. They therefore find use in applications ranging from histological analyses to fluorescence-activated cell sorting (FACS). A limiting factor in such studies is the tendency of the colored dye product to diffuse from the site of cleavage, whereby resolution and utility are decreased. This problem was addressed many years ago for visible dyes with the introduction of bromochloroindolyl conjugates, such as 5-bromo-4-chloro-3-indolyl b-d-galactopyranoside (X-Gal), which, when cleaved, dimerize to form an intensely blue product and precipitate; thus, diffusion is minimized. Few such systems have been developed for fluorescent reagents, largely because their precipitation results in fluorescence quenching. An alternative strategy for the reduction of product diffusion would be for the enzyme to release a highly reactive fluorescent species that could covalently derivatize nucleophilic sites in the nearby medium. Sitespecific immobilization of the fluorophore would result, without precipitation, and quenching would therefore be minimized. For example, tyramide signal amplification (TSA) takes advantage of this strategy. Herein, we report the development of simply modified derivatives of coumarin glycosides that are not innately fluorescent, but when cleaved release a fluorescent aglycone that readily forms a quinone methide, which rapidly reacts with a nearby nucleophile. Their use in histological studies and in FACS sorting of cell types is demonstrated. Enzyme substrates that generate latent quinone methides have been developed in the past for a range of enzymes, including proteases, esterases, phosphatases, sulfatases, and glycosidases. The most commonly employed are those containing orthoor para-(di)halomethyl phenols, which, when liberated, generate reactive quinone methides (Scheme 1). These compounds were originally developed as mechanism-based inactivators for the selective inhibition of specific activities or the labeling and identification of activesite residues. However, the time taken for the (di)halomethyl phenol to decompose, and for the quinone methide thereby generated to react, is often sufficiently long for the reagent to leave the active site and react with other nearby nucleophiles, including water. This problem is further aggravated when, as in most cases, the aryl moiety has no specific affinity for the active site. This behavior has significantly reduced the utility of such compounds and, despite numerous reports, has rendered them essentially useless as probes for activity-based proteomics studies, since, although generated by the specific enzyme activity, the quinone methides react indiscriminately. However, this behavior is ideal for imaging agents of the type proposed. Indeed, the perfect reagent would never react at the active site, since that may inactivate the enzyme, but rather would react on the exterior of the protein, or with cellular components in the immediate vicinity. To test this approach, we synthesized glycosides 1, 2, and 3 (Scheme 1). The choice of the difluoromethyl over the monofluoromethyl derivative was based upon the greater stability of the parent compound towards solvolysis and upon the longer anticipated “lifetime” of the dihalomethyl phenol; the longer lifetime improves its chances of diffusing out of the active site. The simple synthetic route employed (see the Experimental Section and the Supporting Information) could be Scheme 1. A) Generation of and nucleophilic addition to a quinone methide. B) Fluorogenic quinone methide generating glycoside substrates synthesized in this study.

Thioglycosynthases: double mutant glycosidases that serve as scaffolds for thioglycoside synthesis

A double mutant, retaining glycosidase that lacks both the catalytic nucleophile and the catalytic acid/base residues efficiently catalyzes thioglycoside formation from a glycosyl fluoride donor and thiosugar acceptors.

Thermostable Glycosynthases and Thioglycoligases Derived from Thermotoga maritima β-Glucuronidase

Uronic acid-containing glycoconjugates are found in a number of different and important contexts. Examples include pectins and hemicelluloses within plant cell walls, glycosaminoglycans (GAGs) within the mammalian extracellular matrix, capsular poly ACHTUNGTRENNUNGsaccharides of bacteria and glucuronide conjugates formed as a means of solubilisation and clearance of unwanted molecules. Of these, GAGs such as heparin, heparan sulfate, chondroitin sulfate and hyaluronan are of particular importance in a variety of biological processes, and analogues of these structures might well be useful as therapeutic agents. 2] While the chemical syntheses of short oligosaccharide fragments of GAGs have been achieved, these syntheses are surprisingly challenging, and their scale-up to the levels needed for clinical trials remains challenging. An alternative synthetic approach involves the use of enzymes, and, in that regard, ACHTUNGTRENNUNGadvances have been made in several areas. The enzymes ACHTUNGTRENNUNGinvolved in GAG biosynthesis, the glycosyltransferases, have proved problematic, largely because they are generally closely membrane-associated. However, considerable success has been achieved by DeAngelis’ group with hyaluronan synthase, and small-scale syntheses with recombinant enzymes are now available. 5] Glycosidases run “in reverse” provide the other approach, and, indeed, Kobayashi et al. have successfully assembled hyaluronan and chondroitin sulfate oligosaccharides from disaccharide precursors, converted into their oxazolines, by use of endo-hexosaminidases. 7] An alternative strategy for oligosaccharide assembly involves the use of retaining glycosidases in which either the catalytic nucleophile (glycosynthases) or the acid/base catalyst (thioACHTUNGTRENNUNGglycoligases) is mutated (Scheme 1). Glycosynthases are hydroACHTUNGTRENNUNGlytically incompetent mutants that can, nevertheless, effect efficient glycosyl transfer from a glycosyl fluoride sugar donor of opposite anomeric configuration to that of the natural substrate. The glycosyl fluoride donor binds and acts as a mimic of the normal glycosyl enzyme. A range of such enzymes has now been produced, and directed evolution has generated highly efficient catalysts (kcat 90 s ). Thioglycoligases carry out glycosyl transfer from an activated glycosyl donor of normal configuration to a thiosugar acceptor, with formation of a thioglycosidic linkage. In this case, the activated leaving group “complements” the absence of the acid catalyst in the formation of the glycosyl enzyme, while the highly nucleophilic thiol ACHTUNGTRENNUNG(ate) “complements” the missing general base catalyst. While a range of glycosynthases and thioglycoligases has now been produced, and directed evolution approaches have been employed to boost rates and alter specificities, there have been no reports to date of either glycosynthases or thioglycoligases that transfer glycuronyl residues. Given the importance of these structures and the difficulties noted earlier with effective synthesis, we investigated the potential for both classes of mutant enzymes in the synthesis of glycuronyl linkages. An appropriate candidate glycuronidase for the generation of both a glycosynthase and a thioglycoligase was the thermostable b-glucuronidase of Thermotoga maritima (TMGUA), a member of GH family 2 (http://afmb.cnrs-mrs.fr/ CAZY/), particularly as the identities of both the nucleophile (E476) and the acid/base catalyst (E383) have recently been confirmed.

Observing cellulose biosynthesis and membrane translocation in crystallo

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA–BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate- and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a ‘finger helix’ that contacts the polymer’s terminal glucose. Cooperating with BcsA’s gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. This mechanism is validated experimentally by tethering BcsA’s finger helix, which inhibits polymer translocation but not elongation.

A β-1,4-Galactosyltransferase from Helicobacter pylori is an Efficient and Versatile Biocatalyst Displaying a Novel Activity for Thioglycoside Synthesis

Helicobacter pylori is a highly persistent and common pathogen in humans. It is the causative agent of chronic gastritis and its further stages. HP0826 is the β‐1,4‐galactosyltransferase involved in the biosynthesis of the LPS O‐chain backbone of H. pylori. Though it was first cloned nearly a decade ago, there are surprisingly limited data about the characteristics of HP0826, especially given its prominent role in H. pylori pathogenicity. We here demonstrate that HP0826 is a highly efficient and promiscuous biocatalyst. We have exploited two novel enzymatic activities for the quantitative synthesis of the thiodisaccharide Gal‐β‐S‐1,4‐GlcNAc‐pNP as well as Gal‐β‐1,4‐Man‐pNP. We further show that Neisseria meningitidis β‐1,4‐galactosyltransferases LgtB can be used as an equally efficient catalyst in the latter reaction. Thiodisaccharides have been extensively used in structural biology but can also have therapeutic uses. The Gal‐β‐1,4‐Man linkage is found in the Leishmania species LPG backbone disaccharide repeats and cap, which have been associated with vector binding in Leishmaniasis.

Thioglycoligase-Based Assembly of Thiodisaccharides: Screening as β-Galactosidase Inhibitors

Carbohydrates in cells play very important roles in a wide range of biological processes, and impact health and disease.[1] Consequently, interference with the recognition and processing of carbohydrates is a strategy for drug development that is gaining favour. Examples of drugs that are already in the clinic include amylase inhibitors for the reduction of blood glucose levels and sialidase inhibitors as anti-influenza drugs.[2] A newer class of molecules currently under development are those that stabilise otherwise unstable mutant forms of lysosomal glycosidases, and thereby chaperone them to their lysosomal location and bypass proteasomal degradation. These pharmacological chaperones are typically inhibitors, but can be used to rescue deficient glycosidase activity in lysosomes and thereby provide a potential treatment for this class of storage disorders.[3] The glycosidase inhibitors employed in such approaches are typically high affinity transition-state analogues, but such compounds often do not have high linkage specificity for the specific target glycosidase since they typically do not contain components of the “aglycone”. Further, very high affinities are not desirable in this application since potent inhibitors would not be released upon reaching the lysosome. Alternatively, noncleavable substrate analogues can be used as competitive inhibitors for both glycosidases and carbohydrate-binding proteins. Although typically less potent, such reagents are generally more specific. Some of the best such analogues are thioglycosides, wherein a sulfur atom replaces the intersugar glycosidic oxygen of the normal substrate. These are generally good mimics of the natural substrate, but are recalcitrant to cleavage by essentially all glycosidases.[4]

Facile Synthesis of 2,4‐Dinitrophenyl α‐D‐Glycopyranosides as Chromogenic Substrates for α‐Glycosidases

Amongst the chromogenic substrates used to assay glycosidases, one of the most useful classes is that of the 2,4-dinitrophenyl glycosides, which were originally introduced by Capon and Thomson. Their particular value lies in the low pKa of the 2,4-dinitrophenol leaving group ( 4.0), which endows the substrate with high reactivity, thus high hydrolytic rates if glycoside bond fission is the ratelimiting step. Further, the low pKa of the phenol allows continuous assay even at relatively low pH values. This is particularly important for assay and kinetic analysis of lysosomal glycosidases, whose optimum pH is typically around pH 4–5. While synthesis of most aryl a-glycosides can be achieved by conventional approaches that involve reaction of the phenol, or phenolate, with an activated sugar derivative, this approach is particularly challenging for aN-acetylhexosaminides. Furthermore, the low nucleophilicity of this particular phenol renders this approach troublesome and indeed unsuccessful. An alternative approach to the synthesis of 2,4-dinitrophenyl glycosides, which was introduced by Van Boom et al. , is to treat the partially protected sugar hemiacetal with 1-fluoro-2,4-dinitrobenzene in the presence of a hindered base, typically 1,4-diazabicyclo ACHTUNGTRENNUNG[2.2.2]octane (DABCO). This nucleophilic aromatic substitution reaction provides good yields of the equatorial (generally b) glycoside— the kinetic product. This can then be converted to the a-glycoside by treatment with a stronger base, such as potassium carbonate in solvents like DMF or DMSO. A mechanism for this isomerisation reaction, which involves nucleophilic aromatic substitution, was demonstrated by Berven et al. 6] A need for substrates for the study of a-N-acetylhexosaminidases recently led us to attempt to synthesise the corresponding 2,4-dinitrophenyl a-N-acetylhexosaminides with this approach. Unfortunately, the extreme lability of the aryl b-hexosaminide intermediate due to neighbouring group participation from the amide, rendered this route useless. Consequently, we speculated that a one-step synthesis that involves the use of a strong base to catalyse both the formation and isomerisation of the b-glycoside might prove to be a better approach since the first-formed b-glycoside intermediate might well undergo isomerisation more rapidly than decomposition. Furthermore, by using a slight excess of 1-fluoro-2,4-dinitrobenzene any ACHTUNGTRENNUNGdecomposed intermediates could be reconverted into product, in situ. In the expectation that substantial decomposition of the intermediate 2,4-dinitrophenyl b-glycosides of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) would occur, a twofold excess of 1-fluoro-2,4-dinitrobenzene was initially employed by using potassium carbonate as base catalyst in dimethylformamide (DMF; Scheme 1). Under these conditions the desired, fully acetylated 2,4-dinitrophenyl a-glycoside was formed in each case, but was accompanied by substantial

Catalytic properties of a mutant β-galactosidase from Xanthomonas manihotis engineered to synthesize galactosyl-thio-β-1,3 and -β-1,4-glycosides

The identity of the acid/base catalyst of the Family 35 β‐galactosidases from Xanthomonas manihotis (BgaX) has been confirmed as Glu184 by kinetic analysis of mutants modified at that position. The Glu184Ala mutant of BgaX is shown to function as an efficient thioglycoligase, which synthesises thiogalactosides with linkages to the 3 and 4 positions of glucosides and galactosides in high (>80%) yields. Kinetic analysis of the thioglycoligase reveals glycosyl donor K m values of 1.5–21 μM and glycosyl acceptor K m values from 180 to 500 μM. This mutant should be a valuable catalyst for the synthesis of metabolically stable analogues of this important glycosidic linkage.