All-in-one assay for β-d-galactoside sialyltransferases: Quantification of productive turnover, error hydrolysis, and site selectivity

Czabany, Tibor; Schmölzer, Katharina; Luley‐Goedl, Christiane; Ribitsch, Doris; Nidetzky, Bernd

doi:10.1016/j.ab.2015.04.035

Cited by 9 publications

(12 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The high glycosylation efficiency with Leloir GTs arises from a favorable thermodynamic equilibrium K eq in these examples, determined by the sugar nucleotide donor and carbohydrate or aglycone acceptor, pH, and ionic strength. As mentioned earlier, in a few examples the hydrolysis of the NDP-sugar donor has been reported for Leloir GTs [70,71,72,73,74,75,76,77]. In these particular cases, it is important to emphasize that the glycosylation with Leloir GTs is under kinetic control, and the sugar acceptor and water are competing nucleophiles throughout the entire course of reaction [70,71,72,73,74,75,76,77].…”

Section: Application Of Glycosyl Transferases In Organic Synthesismentioning

confidence: 95%

“…The exclusion of hydrolysis activity of the nucleotide sugar donor separates glycoside hydrolases from glycosyltransferases. Nevertheless, hydrolysis of the nucleotide sugar donor in the absence of a sugar acceptor has been reported and is referred to as “error hydrolysis” [70,71,72,73]. Hence, the competition between water or a sugar acceptor as nucleophile is important for the efficiency of glycosylation.…”

Section: Glycosyltransferases In Naturementioning

confidence: 99%

See 1 more Smart Citation

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

Mestrom

Przypis

Kowalczykiewicz

et al. 2019

IJMS

View full text Add to dashboard Cite

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio-and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.hydroxynitrile lyases catalyzed the enzymatic hydrolysis of the glycoside amygdalin [3]. Moving almost two centuries forward, the largest volumetric biocatalytic industrial process is the application of glucose isomerase for the production of high fructose syrup for food and drink applications, producing fructose from glucose at 10 7 tons per year [4]. The secret of the success of enzymes in the production or treatment of carbohydrates and glycosides is their exquisite stereo-and regioselectivity. The excellent selectivity of enzymes is required due to the diversity of structural features of carbohydrates [5], comprising d-and l-epimers, ring size, anomeric configuration, linkages, branching, and oxidation state(s). Since drug targets often exhibit specificity for all of these structural features, the production process should not contain any side-products to prevent undesired side-effects [6].The challenge in the synthesis of carbohydrates is their wide variety of functionalities and stereochemistry ( Figure 1). (Poly)hydroxyaldehydes containing a terminal aldehyde are referred to as aldoses and (poly)hydroxyketones are defined as ketoses. In aqueous solutions, monosaccharides form equilibrium mixtures of linear open-chain and ring-closed 5-or 6-membered furanoses or pyranoses, respectively. For aldoses, the asymmetric ring forms at C-1. For ketoses, it closes at C-2 as an axial (α) or equatorial (β) hemiacetal or hemiketal, respectively (commonly defined as the anomeric center). A glycosidic linkage is a covalent O-, S-, N-, or C-bond connecting a monosaccharide to another residue resulting in a glycoside, while glucoside is specific for a glucose moiety. The equatorial or axial position of the glycosidic bond is referred to as α-(axial) or β-linkage (equatorial). The number of carbohydrates linked via glycosidic bonds can be subdivided into oligosaccharides with two to ten linked carbohydrates, while polysaccharides (glycans) contain more than ten glycosidic bonds. A glycan either con...

show abstract

Section: Application Of Glycosyl Transferases In Organic Synthesismentioning

confidence: 95%

Section: Glycosyltransferases In Naturementioning

confidence: 99%

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

Mestrom

Przypis

Kowalczykiewicz

et al. 2019

IJMS

View full text Add to dashboard Cite

show abstract

Section: Resultsmentioning

confidence: 99%

“…Most methods for assaying sialyltransferase activity reported so far are discontinuous and involve laborious quenching of the catalytic reaction by acido rh eating followed by product analysis. [36,38,42] HPLC analysiso ff luorescently labeled substrates [33] is indirect, and surrogate substrates may show ak inetic behavior different from the native substrates. [43] The only continuous assay for SiaT activity is based on the indirect measurement of CMP release by an enzymatic link to NADPH consumption, which is inappropriate for discrimination of sialidase activity.…”

Section: Activityscreening and Hit Characterizationmentioning

confidence: 99%

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Mertsch

Dong

et al. 2020

Chemistry A European J

View full text Add to dashboard Cite

Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaTpph) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure‐guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaTpph (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3‐Neu5Ac‐Lac) from lactose in a one‐pot, two‐enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new‐to‐nature sialoconjugates for biological studies.

show abstract

“…In 1998, Schmidt and co‐workers introduced an HPLC‐based assay using the p ‐nitrophenyl glycoside of N ‐acetyllactosamine as a UV‐labeled acceptor, and this continues to be one of the most widely used ST inhibition assays today . Recent extensions of this method, include a continuous all‐in‐one HPLC‐based assay that enables the quantification of productive turnover, error hydrolysis, and site selectivity of sialyltransfer and donor substrate hydrolysis, with the potential for HTS . Further HPLC‐based assays include those reported by Kijahara et al.…”

Section: Biological Assaysmentioning

confidence: 99%

Advancement of Sialyltransferase Inhibitors: Therapeutic Challenges and Opportunities

Szabo

Skropeta

2016

Medicinal Research Reviews

View full text Add to dashboard Cite

Hypersialylation of tumor cell surface proteins along with a marked upregulation of sialyltransferase (ST) activity is a well-established hallmark of cancer. Due to the critical role of STs in tumor growth and progression, ST inhibition has emerged as a potential new antimetastatic strategy for a range of cancers including pancreatic and ovarian. Human STs are divided into subtypes based on their linkage and acceptor molecule, with each subtype controlling the synthesis of specific sialylated structures with unique biological roles. This has important implications for inhibitor development, as STs also play significant roles in immune responses, inflammation, viral infection, and neurological disorders. Thus, the current goal in order to advance to the clinic is the development of subtype selective, cell-permeable and synthetically accessible, smallmolecule ST inhibitors. Herein is a comprehensive review of the latest developments in ST inhibitors from design, Nature, and high-throughput screening, addressing both the challenges and opportunities in targeting cell surface sialylation. The review features an overview of the biological evaluation methods, computational and imaging tools, inhibitor molecular diversity, and selectivity toward ST subtypes, along with the emerging role of ST inhibitors as diagnostic tools for disease imaging.

show abstract

All-in-one assay for β-d-galactoside sialyltransferases: Quantification of productive turnover, error hydrolysis, and site selectivity

Cited by 9 publications

References 55 publications

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Advancement of Sialyltransferase Inhibitors: Therapeutic Challenges and Opportunities

Contact Info

Product

Resources

About