An alternative broad-specificity pathway for glycan breakdown in bacteria.

The vast majority of glycosidases characterized to date follow one of the variations of the 'Koshland' mechanisms1 to hydrolyse glycosidic bonds through substitution reactions. Here we describe a large-scale screen of a human gut microbiome metagenomic library using an assay that selectively identifies non-Koshland glycosidase activities2. Using this, we identify a cluster of enzymes with extremely broad substrate specificities and thoroughly characterize these, mechanistically and structurally. These enzymes not only break glycosidic linkages of both α and ß stereochemistry and multiple connectivities, but also cleave substrates that are not hydrolysed by standard glycosidases. These include thioglycosides, such as the glucosinolates from plants, and pseudoglycosidic bonds of pharmaceuticals such as acarbose. This is achieved through a distinct mechanism of hydrolysis that involves oxidation/reduction and elimination/hydration steps, each catalysed by enzyme modules that are in many cases interchangeable between organisms and substrate classes. Homologues of these enzymes occur in both Gram-positive and Gram-negative bacteria associated with the gut microbiome and other body parts, as well as other environments, such as soil and sea. Such alternative step-wise mechanisms appear to constitute largely unrecognized but abundant pathways for glycan degradation as part of the metabolism of carbohydrates in bacteria.

A high-throughput screening platform for enzymes active on mucin-type O-glycoproteins.

Mucin-type O-glycosylation is a post-translational modification present at the interface between cells where it has important roles in cellular communication. However, deciphering the function of O-glycoproteins and O-glycans can be challenging, especially as few enzymes are available for their assembly or selective degradation. Here, to address this deficiency, we developed a genetically encoded screening methodology for the discovery and engineering of the diverse classes of enzymes that act on O-glycoproteins. The method uses Escherichia coli that have been engineered to produce an O-glycosylated fluorescence resonance energy transfer probe that can be used to screen for O-glycopeptidase activity. Subsequent cleavage of the substrate by O-glycopeptidases provides a read-out of the glycosylation state of the probe, allowing the method to also be used to assay glycosidases and glycosyltransferases. We further show the potential of this methodology in the first ultrahigh-throughput-directed evolution of an O-glycopeptidase.

Vinyl Halide-Modified Unsaturated Cyclitols are Mechanism-Based Glycosidase Inhibitors.

Suitably configured allyl ethers of unsaturated cyclitols act as substrates of ß-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of ß-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages. Structures of complexes with the Sulfolobus ß-glucosidase revealed similar enzyme-ligand interactions to those seen in complexes with a 2-fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7-fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.

Mammalian sialyltransferases allow efficient Escherichia coli-based production of mucin-type O-glycoproteins but can also transfer Kdo.

The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an Escherichia coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein and another O-glycosylation machinery. Here, we expand the capabilities of our platform to carry out sialylation and demonstrate the high-yielding production of human interferon α2b and human growth hormone bearing mono- and disialylated T-antigen glycans. This is achieved through engineering an E. coli strain to produce CMP-Neu5Ac and introducing various α-2,3- and α-2,6 mammalian or bacterial sialyltransferases into our O-glycosylation operons. We further demonstrate that mammalian sialyltransferases, including porcine ST3Gal1, human ST6GalNAc2 and human ST6GalNAc4, are very effective in vivo and outperform some of the bacterial sialyltransferases tested, including Campylobacter jejuni Cst-I and Cst-II. In the process, we came upon a way of modifying T-Antigen with Kdo, using a previously uncharacterised Kdo-transferase activity of porcine ST3Gal1. Ultimately, the heterologous expression of mammalian sialyltransferases in E. coli shows promise for the further development of bacterial systems in therapeutic glycoprotein production.

Comparison of α2,6-sialyltransferases for sialylation of therapeutic proteins.

The development of therapeutic proteins for the treatment of numerous diseases is one of the fastest growing areas of biotechnology. Therapeutic efficacy and serum half-life are particularly important, and these properties rely heavily on the glycosylation state of the protein. Expression systems to produce authentically fully glycosylated therapeutic proteins with appropriate terminal sialic acids are not yet perfected. The in vitro modification of therapeutic proteins by recombinant sialyltransferases offers a promising and elegant strategy to overcome this problem. Thus, the detailed expression and characterization of sialyltransferases for completion of the glycan chains is of great interest to the community. We identified a novel α2,6-sialyltransferase from Helicobacter cetorum and compared it to the human ST6Gal1 and a Photobacterium sp. sialyltransferase using glycoprotein substrates in a 96-well microtiter-plate-based assay. We demonstrated that the recombinant α2,6-sialyltransferase from H. cetorum is an excellent catalyst for modification of N-linked glycans of different therapeutic proteins.

Directed evolution of bacterial polysialyltransferases.

Polysialyltransferases (polySTs) are glycosyltransferases that synthesize polymers of sialic acid found in vertebrates and some bacterial pathogens. Bacterial polySTs have utility in the modification of therapeutic proteins to improve serum half-life, and the potential for tissue engineering. PolySTs are membrane-associated proteins and as recombinant proteins suffer from inherently low solubility, low expression levels and poor thermal stability. To improve their physicochemical and biochemical properties, we applied a directed evolution approach using a FACS-based ultrahigh-throughput assay as a simple, robust and reliable screening method. We were able to enrich a large mutant library and, in combination with plate-based high-throughput secondary screening, we discovered mutants with increased enzymatic activity and improved stability compared to the wildtype enzyme. This work presents a powerful strategy for the screening of directed evolution libraries of bacterial polySTs to identify better catalysts for in vitro polysialylation of therapeutics.

Characterization of a thermostable endoglucanase from Cellulomonas fimi ATCC484.

Bacteria in the genus Cellulomonas are well known as secretors of a variety of mesophilic carbohydrate degrading enzymes (e.g., cellulases and hemicellulases), active against plant cell wall polysaccharides. Recent proteomic analysis of the mesophilic bacterium Cellulomonas fimi ATCC484 revealed uncharacterized enzymes for the hydrolysis of plant cell wall biomass. Celf_1230 (CfCel6C), a secreted protein of Cellulomonas fimi ATCC484, is a novel member of the GH6 family of cellulases that could be successfully expressed in Escherichia coli. This enzyme displayed very little enzymatic/hydrolytic activity at 30 °C, but showed an optimal activity around 65 °C, and exhibited a thermal denaturation temperature of 74 °C. In addition, it also strongly bound to filter paper despite having no recognizable carbohydrate binding module. Our experiments show that CfCel6C is a thermostable endoglucanase with activity on a variety of ß-glucans produced by an organism that struggles to grow above 30 °C.

Convergent evolution of polysaccharide debranching defines a common mechanism for starch accumulation in cyanobacteria and plants.

Starch, unlike hydrosoluble glycogen particles, aggregates into insoluble, semicrystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a preexisting cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semicrystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably, both the plant and cyanobacterial enzymes have evolved through convergent evolution, showing novel yet identical substrate specificities from a preexisting enzyme that originally displayed the much narrower substrate preferences required for glycogen catabolism.

Crystal structure of the Chlamydomonas starch debranching enzyme isoamylase ISA1 reveals insights into the mechanism of branch trimming and complex assembly.

The starch debranching enzymes isoamylase 1 and 2 (ISA1 and ISA2) are known to exist in a large complex and are involved in the biosynthesis and crystallization of starch. It is suggested that the function of the complex is to remove misplaced branches of growing amylopectin molecules, which would otherwise prevent the association and crystallization of adjacent linear chains. Here, we investigate the function of ISA1 and ISA2 from starch producing alga Chlamydomonas. Through complementation studies, we confirm that the STA8 locus encodes for ISA2 and sta8 mutants lack the ISA1·ISA2 heteromeric complex. However, mutants retain a functional dimeric ISA1 that is able to partly sustain starch synthesis in vivo. To better characterize ISA1, we have overexpressed and purified ISA1 from Chlamydomonas reinhardtii (CrISA1) and solved the crystal structure to 2.3 Å and in complex with maltoheptaose to 2.4 Å. Analysis of the homodimeric CrISA1 structure reveals a unique elongated structure with monomers connected end-to-end. The crystal complex reveals details about the mechanism of branch binding that explains the low activity of CrISA1 toward tightly spaced branches and reveals the presence of additional secondary surface carbohydrate binding sites.

Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-isomaltase N-terminal domains.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear alpha-1,4- and branched alpha-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear alpha-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both alpha-1,4- and alpha-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 A and in complex with the inhibitor kotalanol to 2.15 A resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic +1 subsite, which may account for its additional substrate specificity for alpha-1,6 substrates.

Mapping the intestinal alpha-glucogenic enzyme specificities of starch digesting maltase-glucoamylase and sucrase-isomaltase.

Inhibition of intestinal α-glucosidases and pancreatic α-amylases is an approach to controlling blood glucose and serum insulin levels in individuals with Type II diabetes. The two human intestinal glucosidases are maltase-glucoamylase and sucrase-isomaltase. Each incorporates two family 31 glycoside hydrolases responsible for the final step of starch hydrolysis. Here we compare the inhibition profiles of the individual N- and C-terminal catalytic subunits of both glucosidases by clinical glucosidase inhibitors, acarbose and miglitol, and newly discovered glucosidase inhibitors from an Ayurvedic remedy used for the treatment of Type II diabetes. We show that features of the compounds introduce selectivity towards the subunits. Together with structural data, the results enhance the understanding of the role of each catalytic subunit in starch digestion, helping to guide the development of new compounds with subunit specific antidiabetic activity. The results may also have relevance to other metabolic diseases such as obesity and cardiovascular disease.

Discovery and Development of Promiscuous O-Glycan Hydrolases for Removal of Intact Sialyl T-Antigen.

Mucin-type O-glycosylation (O-glycosylation) is a common post-translational modification that confers distinct biophysical properties to proteins and plays crucial roles in intercellular signaling. Yet, despite the importance of O-glycans, relatively few tools exist for their analysis and modification. In particular, there is a need for enzymes that can cleave the wide range of O-glycan structures found on protein surfaces, to facilitate glycan profiling and editing. Through functional metagenomic screening of the human gut microbiome, we discovered endo-O-glycan hydrolases from CAZy family GH101 that are capable of slowly cleaving the intact sialyl T-antigen trisaccharide (a ubiquitous O-glycan structure in humans) in addition to their primary activity against the T-antigen disaccharide. We then further explored this sequence space through phylogenetic profiling and analysis of representative enzymes, revealing large differences in the levels of this promiscuous activity between enzymes within the family. Through structural and sequence analysis, we identified active site residues that modulate specificity. Through subsequent rational protein engineering, we improved the activity of an enzyme identified by phylogenetic profiling sufficiently that substantial removal of the intact sialyl T-antigen from proteins could be readily achieved. Our best sialyl T-antigen hydrolase mutant, SpGH101 Q868G, is further shown to function on a number of proteins, tissues, and cells. Access to this enzyme opens up improved methodologies for unraveling the glycan code.

7-Fluorosialyl Glycosides Are Hydrolysis Resistant but Readily Assembled by Sialyltransferases Providing Easy Access to More Metabolically Stable Glycoproteins.

The maintenance of therapeutic glycoproteins within the circulatory system is associated, in large part, with the integrity of sialic acids as terminal sugars on the glycans. Glycoprotein desialylation, either by spontaneous cleavage or through host sialidases, leads to protein clearance, mainly through the liver. Thus, the installation of minimally modified sialic acids that are hydrolysis-resistant yet biologically equivalent should lead to increased circulatory half-lives and improved pharmacokinetic profiles. Here we describe the chemoenzymatic synthesis of CMP-sialic acid sugar donors bearing fluorine atoms at the 7-position, starting from the corresponding 4-deoxy-4-fluoro-N-acetylhexosamine precursors. For the derivative with natural stereochemistry we observe efficient glycosyl transfer by sialyltransferases, along with improved stability of the resultant 7-fluorosialosides toward spontaneous hydrolysis (3- to 5-fold) and toward cleavage by GH33 sialidases (40- to 250-fold). Taking advantage of the rapid transfer of 7-fluorosialic acid by sialyltransferases, we engineered the O-glycan of Interferon α-2b and the N-glycans of the therapeutic glycoprotein α1-antitrypsin. Studies of the uptake of the glyco-engineered α1-antitrypsin by HepG2 liver cells demonstrated the bioequivalence of 7-fluorosialic acid to sialic acid in suppressing interaction with liver cell lectins. In vivo pharmacokinetic studies reveal enhanced half-life of the protein decorated with 7-fluorosialic acid relative to unmodified sialic acid in the murine circulatory system. 7-Fluorosialylation therefore offers considerable promise as a means of prolonging circulatory half-lives of glycoproteins and may pave the way toward biobetters for therapeutic use.

Prevention of vascular-allograft rejection by protecting the endothelial glycocalyx with immunosuppressive polymers.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity. Polymer-mediated shielding of the endothelial glycocalyx following organ procurement should be compatible with clinical procedures for transplant preservation and perfusion, and may reduce the damage and rejection of transplanted organs after surgery.

New glucosidase inhibitors from an ayurvedic herbal treatment for type 2 diabetes: structures and inhibition of human intestinal maltase-glucoamylase with compounds from Salacia reticulata.

An approach to controlling blood glucose levels in individuals with type 2 diabetes is to target alpha-amylases and intestinal glucosidases using alpha-glucosidase inhibitors acarbose and miglitol. One of the intestinal glucosidases targeted is the N-terminal catalytic domain of maltase-glucoamylase (ntMGAM), one of the four intestinal glycoside hydrolase 31 enzyme activities responsible for the hydrolysis of terminal starch products into glucose. Here we present the X-ray crystallographic studies of ntMGAM in complex with a new class of alpha-glucosidase inhibitors derived from natural extracts of Salacia reticulata, a plant used traditionally in Ayuverdic medicine for the treatment of type 2 diabetes. Included in these extracts are the active compounds salacinol, kotalanol, and de-O-sulfonated kotalanol. This study reveals that de-O-sulfonated kotalanol is the most potent ntMGAM inhibitor reported to date (K(i) = 0.03 microM), some 2000-fold better than the compounds currently used in the clinic, and highlights the potential of the salacinol class of inhibitors as future drug candidates.

Probing the active-site requirements of human intestinal N-terminal maltase-glucoamylase: Synthesis and enzyme inhibitory activities of a six-membered ring nitrogen analogue of kotalanol and its de-O-sulfonated derivative.

In order to probe the active-site requirements of the human N-terminal subunit of maltase-glucoamylase (ntMGAM), one of the clinically relevant intestinal enzymes targeted for the treatment of type-2 diabetes, the syntheses of two new inhibitors are described. The target compounds are structural hybrids of kotalanol, a naturally occurring glucosidase inhibitor with a unique five-membered ring sulfonium-sulfate inner salt structure, and miglitol, a six-membered ring antidiabetic drug that is currently in clinical use. The compounds comprise the six-membered ring of miglitol and the side chain of kotalanol or its de-O-sulfonated derivative. Inhibition studies of these hybrid molecules with human ntMGAM indicated that they are inhibitors of this enzyme with comparable K(i) values to that of miglitol (kotalanol analogue: 2.3±0.6µM; corresponding de-O-sulfonated analogue: 1.4±0.5µM; miglitol: 1.0±0.1µM). However, they are less active compared to kotalanol (K(i)=0.19±0.03µM). These results suggest that the (3)T(2) enzyme-bound conformation of the five-membered thiocyclitol moiety of the kotalanol class of compounds more closely resembles the (4)H(3) conformation of the proposed transition state for the formation of an enzyme-substrate covalent intermediate in the glycosidase hydrolase family 31 (GH31)-catalyzed reaction.

Total syntheses of casuarine and its 6-O-alpha-glucoside: complementary inhibition towards glycoside hydrolases of the GH31 and GH37 families.

Total synthesis of naturally occurring casuarine (1) and the first total synthesis of casuarine 6-O-alpha-glucoside (2) were achieved through complete stereoselective nitrone cycloaddition, Tamao-Fleming oxidation and selective alpha-glucosylation as key steps. Biological assays of the two compounds proved their strong and selective inhibitory properties towards glucoamylase NtMGAM and trehalase Tre37A, respectively, which place them among the most powerful inhibitors of these enzymes. The structural determination of the complexes of NtMGAM with 1 and of Tre37A with 2 revealed interesting similarities in the catalytic sites of these two enzymes which belong to different families and clans.

A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans.

We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as well as a ß1,3-galactosyl transferase and UDP-Glc(NAc)-4-epimerase, both from Campylobacter jejuni, and a disulfide bond isomerase of bacterial or human origin. The effectiveness of this two-plasmid synthetic operon system has been tested on three proteins with therapeutic potential: the native and an engineered version of the naturally O-glycosylated human interferon α-2b, as well as human growth hormone with one engineered site of glycosylation. Having established proof of principle for the addition of the core-1 glycan onto proteins, we are now developing this system as a platform for producing and modifying human protein therapeutics with more complex O-glycan structures in E. coli.

An enzymatic pathway in the human gut microbiome that converts A to universal O type blood.

Access to efficient enzymes that can convert A and B type red blood cells to 'universal' donor O would greatly increase the supply of blood for transfusions. Here we report the functional metagenomic screening of the human gut microbiome for enzymes that can remove the cognate A and B type sugar antigens. Among the genes encoded in our library of 19,500 expressed fosmids bearing gut bacterial DNA, we identify an enzyme pair from the obligate anaerobe Flavonifractor plautii that work in concert to efficiently convert the A antigen to the H antigen of O type blood, via a galactosamine intermediate. The X-ray structure of the N-acetylgalactosamine deacetylase reveals the active site and mechanism of the founding member of an esterase family. The galactosaminidase expands activities within the CAZy family GH36. Their ability to completely convert A to O of the same rhesus type at very low enzyme concentrations in whole blood will simplify their incorporation into blood transfusion practice, broadening blood supply.

Luminal starch substrate "brake" on maltase-glucoamylase activity is located within the glucoamylase subunit.

The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.