Successfully Engineering a Bacterial Sialyltransferase for Regioselective α2,6-sialylation

Xu, Yangyang; Fan, Yunge; Ye, Jinfeng; Wang, Faxing; Nie, Quandeng; Wang, Li; Wang, Peng George; Cao, Hongzhi; Cheng, Jiansong

doi:10.1021/acscatal.8b01993

Cited by 28 publications

(24 citation statements)

References 40 publications

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“…The reactions were stopped by adding 10 μl TFA and analyzed by HPLC for the formation of products. The data were fitted into the Michaelis–Menten equation using Grafit 6.0 (Erithacus Software; Xu et al ., ).…”

Section: Methodsmentioning

confidence: 97%

Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP‐β‐l‐rhamnose and rhamnosylation mechanism

et al. 2019

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Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP-b-L-rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDPb-L-rhamnose. The mutant H357Q exhibited activity with both UDP-b-L-rhamnose and UDP-glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor-His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism.

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Section: Methodsmentioning

confidence: 97%

Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP‐β‐l‐rhamnose and rhamnosylation mechanism

et al. 2019

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“…The a-(2,6)-sialosides found at the terminal or internal D-galactosyl unit of numerous glycan sequences are generally introduced in humans by two b-galactoside a-(2,6)-sialyltransferases (SiaTs) that have a narrow substrate specificity. Conversely, bacterial a-(2,6)-SiaTs such as b-galactoside a-(2,6)-SiaT from Photobacterium damselae (Pd2,6ST) have been widely used to produce sialosides as they have a more relaxed substrate specificity that could be further extended by enzyme engineering [66]. Combining a structure and sequence analysis, two non-conserved amino acid residues (A200 and S232) found at the bottom of the active site of Pd2,6ST, and potentially differentiating D-Gal/D-GalNAc moieties from the glycan acceptor, were targeted by site-directed mutagenesis to reshape the binding pocket.…”

Section: Synthesis Of Cell-surface Glycansmentioning

confidence: 99%

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

et al. 2019

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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.

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“…A double mutant A200Y/S232Y with bulkier amino acid residues was found to successfully block its binding to internal Gal or GalNAc residues while retain its α2-6-sialylation activity towards terminal Gal or GalNAc sites. A number of monosialylated products were successfully obtained in high yields without the formation of multi-sialylated compounds [62].…”

Section: Enzyme Engineering For Altered Glycosylation Sequence or Impmentioning

confidence: 99%

Strategies for chemoenzymatic synthesis of carbohydrates

McArthur

Chen

2019

Carbohydrate Research

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Carbohydrates are structurally complex but functionally important biomolecules. Therefore, they have been challenging but attractive synthetic targets. While substantial progress has been made on advancing chemical glycosylation methods, incorporating enzymes into carbohydrate synthetic schemes has become increasingly practical as more carbohydrate biosynthetic and metabolic enzymes as well as their mutants with synthetic application are identified and expressed for preparative and large-scale synthesis. Chemoenzymatic strategies that integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions have been extremely powerful. Briefly summarized here are our experiences on developing one-pot multienzyme (OPME) systems and representative chemoenzymatic strategies from others using glycosyltransferase-catalyzed reactions for synthesizing diverse structures of oligosaccharides, polysaccharides, and glycoconjugates. These strategies allow the synthesis of complex carbohydrates including those containing naturally occurring carbohydrate postglycosylational modifications (PGMs) and nonnatural functional groups. By combining these strategies with facile purification schemes, synthetic access to the diverse space of carbohydrate structures can be automated and will not be limited to specialists.

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Successfully Engineering a Bacterial Sialyltransferase for Regioselective α2,6-sialylation

Cited by 28 publications

References 40 publications

Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP‐β‐l‐rhamnose and rhamnosylation mechanism

Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP‐β‐l‐rhamnose and rhamnosylation mechanism

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

Strategies for chemoenzymatic synthesis of carbohydrates

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