Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

Groeve, Manu R.M. De; Remmery, Laurens; Hoorebeke, A. Van; Stout, Jan; Desmet, Tom; Savvides, Savvas N.; Soetaert, Wim

doi:10.1002/bit.22818

Cited by 29 publications

(24 citation statements)

References 25 publications

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“…which is particularly valuable for glycoside synthesis [147]. To further expand the acceptor specificity, De Groeve et al [149] identified a residue (E649) in the structure of CP from C. uda, whose side chain has hydrogen bonding with the D-Glc acceptor. Upon saturation mutagenesis of this residue, the mutant E649C was identified as being able to recognize new glucoside acceptors.…”

Section: Production Of Oligosaccharidesmentioning

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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Section: Production Of Oligosaccharidesmentioning

confidence: 99%

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

et al. 2019

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“…A robust high-throughput screening platform is a prerequisite for targeted development of glycoside phosphorylases with altered substrate specificities to be employed in the synthesis of new glycosides. A triple mutant, containing both donor (T508I/N667A) and acceptor (E649C) mutations, was used as the starting point for creation of more promiscuous enzymes using semi-rational design and site-saturation mutagenesis.Two residues near the entrance of the active site, Asn-156 and Asn-163 were identified to define specificity towards anomerically substituted acceptors.While the wildtype can not glycosylate these acceptors at all, N156D/N163D/T508I/E649C/N667A showed activity towards alkyl-and aryl-β-glucosides and βlinked disaccharides, including β-cellobiosides whose glycosylated derivatives are of interest as novel surfactants, cellulose inhibitors or prebiotic sugars [94].…”

Section: Cellobiose Phosphorylasementioning

confidence: 99%

Carbohydrate synthesis by disaccharide phosphorylases: Reactions, catalytic mechanisms and application in the glycosciences

Luley‐Goedl

Nidetzky

2010

Biotechnology Journal

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Disaccharide phosphorylases are glycosyltransferases (EC 2.4.1.α) of specialized carbohydrate metabolism in microorganisms. They catalyze glycosyl transfer to phosphate using a disaccharide as donor substrate. Phosphorylases for the conversion of naturally abundant disaccharides including sucrose, maltose, α,α‐trehalose, cellobiose, chitobiose, and laminaribiose have been described. Structurally, these disaccharide phosphorylases are often closely related to glycoside hydrolases and transglycosidases. Mechanistically, they are categorized according the stereochemical course of the reaction catalyzed, whereby the anomeric configuration of the disaccharide donor substrate may be retained or inverted in the sugar 1‐phosphate product. Glycosyl transfer with inversion is thought to occur through a single displacement‐like catalytic mechanism, exemplified by the reaction coordinate of cellobiose/chitobiose phosphorylase. Reaction via configurational retention takes place through the double displacement‐like mechanism employed by sucrose phosphorylase. Retaining α,α‐trehalose phosphorylase (from fungi) utilizes a different catalytic strategy, perhaps best described by a direct displacement mechanism, to achieve stereochemical control in an overall retentive transformation. Disaccharide phosphorylases have recently attracted renewed interest as catalysts for synthesis of glycosides to be applied as food additives and cosmetic ingredients. Relevant examples are lacto‐N‐biose and glucosylglycerol whose enzymatic production was achieved on multikilogram scale. Protein engineering of phosphorylases is currently pursued in different laboratories with the aim of broadening the donor and acceptor substrate specificities of naturally existing enzyme forms, to eventually generate a toolbox of new catalysts for glycoside synthesis.

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“…where G n denotes a β-glucan oligomer of length n (n ≥ 2), and G n + 1 denotes a β-glucan oligomer of length n + 1. Although CBP and CDP belong to the same glycoside hydrolase family, they have different substrate specificities 13, 14 .…”

Section: Introductionmentioning

confidence: 99%

Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity

Mao

Fan

et al. 2017

Sci Rep

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A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

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Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

Cited by 29 publications

References 25 publications

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides

Carbohydrate synthesis by disaccharide phosphorylases: Reactions, catalytic mechanisms and application in the glycosciences

Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity

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