Prospecting for microbial α-N-acetylgalactosaminidases yields a new class of GH31 O-glycanase.

α-Linked GalNAc (α-GalNAc) is most notably found at the nonreducing terminus of the blood type-determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylgalactosaminidases are known. Here, to discover and characterize novel microbial enzymes that hydrolyze α-GalNAc, we screened small-insert libraries containing metagenomic DNA from the human gut microbiome. Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that specifically cleaves the initial α-GalNAc from mucin-type O-glycans. This represents a new activity in this GH family and a potentially useful new enzyme class for analysis or modification of O-glycans on protein or cell surfaces.

Chemoenzymatic Approach for the Preparation of Asymmetric Bi-, Tri-, and Tetra-Antennary N-Glycans from a Common Precursor.

Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manß(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the ß-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and ß-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal ß-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric bi- and triantennary glycans could be obtained by removal of a terminal ß-GlcNAc moiety by treatment with ß-N-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary N-glycan found in human breast carcinoma tissue, which represents the most complex N-glycan ever synthesized. Multistage mass spectrometry of the two isomeric triantennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples.

Protein redesign by learning from data.

Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5-45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.

Optimization of inulinase production by a newly isolated Aspergillus tubingensis CR16 using low cost substrates.

Production of an extracellular, thermostable inulinase was carried out by a newly isolated strain of Aspergillus tubingensis CR16 using wheat bran and corn steep liquor (CSL) under solid state fermentation (SSF). Response surface methodology (RSM) involving Box Behnken design (BBD) was employed for the optimization of process parameters viz. time period of fermentation, % moisture content, inoculum size and pH of the medium. Maximum yield of inulinase was 257±11.4 U/g, obtained by inoculating 5 g of wheat bran with 10(9) spores/ml, at initial 71.2% moisture content and pH 6.1 after 103 h of fermentation along with 1358.6±0.8 U/g of invertase activity. Crude inulinase showed maximum activity at 60 °C and pH 5.0. The enzyme was found to be thermostable retaining about 90% of its activity for 4.5 h at 60 °C. Fructose was produced as an end product of inulin hydrolysis proving that the enzyme produced was exoinulinase.

Structural explanation for the acquisition of glycosynthase activity.

Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0-3.0 A from the NW in the wild type. Their positions exhibited a strong correlation with the strength of glycosynthase activity. Here, we propose that a structural prerequisite for the sufficient glycosynthase reaction is the presence of a water molecule at the NW position, and mutation at the NW holder provides a general strategy for inverting GHs. The idea on the position of a water molecule may also be applicable to the design of efficient glycosynthases from retaining GHs.

Artificial enzymes, "chemzymes": current state and perspectives.

Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well as chemzymes that catalyze conjugate additions, cycloadditions, and self-replicating processes. The focus will be mainly on cyclodextrin-based chemzymes since they have shown to be good candidate structures to base an enzyme model skeleton on. In addition hereto, other molecules that encompass binding properties will also be presented.

Facile synthesis toward the construction of an activity probe library for glycosidases.

Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-related research. We have previously demonstrated the design and synthesis of an activity probe for beta-glucosidase adopting a cassette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based on the structure of a key intermediate. The recognition head of this library covers a series of six sugars, including alpha- and beta-d-Glc, alpha- and beta-d-Gal, alpha-d-Man, and alpha-l-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the first time to target alpha-d-glucosidase, alpha-d-galactosidase, and alpha-l-fucosidase, respectively.

Synthesis of the glycosphingolipid beta-galactosyl ceramide and analogues via olefin cross metathesis.

The preparation of the glycosphingolipid galactosyl ceramide from an orthogonally protected five-carbon building block is described. The main chain of the lipid is installed via a highly stereoselective olefin cross metathesis reaction. The methodology permits the facile preparation of glycolipids which vary in the length of the main carbon chain.

An original chemoenzymatic route for the synthesis of beta-D-galactofuranosides using an alpha-L-arabinofuranosidase.

DGalactofuranose is a widespread component of cell wall polysaccharides in bacteria, protozoa and fungi, but is totally absent in mammals. Importantly, galactofuranose is a key constituent of major cell envelope polysaccharides in pathogenic mycobacteria. In this respect, galactofuranose-based glycoconjugates are interesting target molecules for drug design. O-Glycosidases and notably beta-D-galactofuranosidases could be useful tools for the chemoenzymatic synthesis of galactofuranosides, but to date no studies of this type have been reported. Here we report the use of a GH 51 alpha-l-arabinofuranosidase for the synthesis of beta-D-galactofuranosides. We have demonstrated that this enzyme can catalyse both the autocondensation of p-nitrophenyl-beta-D-galactofuranoside and the transgalactofuranosylation of benzyl alpha-D-xylopyranoside, forming p-nitrophenyl beta-D-galactofuranosyl-(1-->2)-beta-D-galactofuranoside and benzyl beta-D-galactofuranosyl-(1-->2)-alpha-D-xylopyranoside, respectively. Both reactions were very regiospecific and the reaction involving benzyl alpha-D-xylopyranoside afforded very high yields (74.8%) of the major product. To our knowledge, this demonstration of chemoenzymatic synthesis of galactofuranosides constitutes the very first use of an O-glycosidase for the synthesis of galactofuranosides.

Facile synthesis of flavonoid 7-O-glycosides.

Highly regioselective removal of the 7-O-acyl groups of the peracylated flavones, isoflavones, and flavonols (PhSH, imidazole, NMP) followed by effective glycosylation with glycosyl trifluoroacetimidates (BF(3).Et(2)O) and cautious deprotection of the acyl groups under basic conditions afforded the desired 7-O-flavonoid glycosides in satisfactory yields.

Enzymes from extremophiles.

The industrial application of enzymes that can withstand harsh conditions has greatly increased over the past decade. This is mainly a result of the discovery of novel enzymes from extremophilic microorganisms. Recent advances in the study of extremozymes point to the acceleration of this trend. In particular, enzymes from thermophilic organisms have found the most practical commercial use to date because of their overall inherent stability. This has also led to a greater understanding of stability factors involved in adaptation of these enzymes to their unusual environments.

Isotope edited NMR studies of glycosidases: design and synthesis of a novel glycosidase inhibitor.

N-13C-methyl-deoxynojirimycin was synthesized and used in isotope-edited NMR studies to probe the binding site of an alpha-glucosidase. Results from this analysis led to the design and preparation of a novel alpha-glucosidase inhibitor, N-glycyl deoxynojirimycin.

Immunochemical localization of heparanase in mouse and human melanomas.

Heparanase, an endo-beta-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a Mr approximately 97,000 protein on SDS-PAGE of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, human A375-SM and mouse B 16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse lungs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells. Our studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; and (c) heparanase antigens are enriched in invasive and metastatic murine and human melanomas in vivo.