696 Send Orders for Reprints to [email protected] Protein & Peptide Letters, 2017, 24, 696-709 REVIEW ARTICLE ISSN: 0929-8665 eISSN: 1875-5305 Impact Factor: 1.068 Glycan Phosphorylases in Multi-Enzyme Synthetic Processes BENTHAM Editor-in-Chief: SCIENCE Ben M. Dunn Giulia Pergolizzia, Sakonwan Kuhaudomlarpa, Eeshan Kalitaa,b and Robert A. Fielda,* aDepartment of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK; bDepartment of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur, Assam -784028, India Abstract: Glycoside phosphorylases catalyse the reversible synthesis of glycosidic bonds by glyco- A R T I C L E H I S T O R Y sylation with concomitant release of inorganic phosphate. The equilibrium position of such reac- tions can render them of limited synthetic utility, unless coupled with a secondary enzymatic step Received: January 17, 2017 Revised: May 24, 2017 where the reaction lies heavily in favour of product. This article surveys recent works on the com- Accepted: June 20, 2017 bined use of glycan phosphorylases with other enzymes to achieve synthetically useful processes. DOI: 10.2174/0929866524666170811125109 Keywords: Phosphorylase, disaccharide, α-glucan, cellodextrin, high-value products, biofuel. O O 1. INTRODUCTION + HO OH Glycoside phosphorylases (GPs) are carbohydrate-active GH enzymes (CAZymes) (URL: http://www.cazy.org/) [1] in- H2O O GP volved in the formation/cleavage of glycosidic bond together O O GT O O + HO O + HO with glycosyltransferase (GT) and glycoside hydrolase (GH) O -- NDP OPO3 NDP -- families (Figure 1) [2-6]. GT reactions favour the thermody- HPO4 namically more stable glycoside product [7]; however, these GS R enzymes can be challenging to work with because of their O O + HO current limited availability and relative instability, along R with the expense of sugar nucleotide substrates [7]. GHs R = F, OpNP thermodynamically favour the hydrolysis of glycosidic bonds; however, active site mutation has allowed their trans- formation into glycosynthases (GSs), which use activated Figure 1. Enzymes involved in glycosidic bond formation and sugars (such as fluoro or p-nitrophenyl glycosides) to drive cleavage. GT = glycosyltransferase; GP = glycoside phosphorylase; reaction in the synthetic direction [7, 8]. GH = glycoside hydrolase; GS = glycoside synthase; NDP = nu- cleotide diphosphate; OpNP = O-para-nitrophenyl. In contrast to GTs and GHs, the equilibrium position for GP reactions tends to result in a mix of starting materials and products, rendering them of modest use in a synthetic sense. glycosidic bond cleavage is similar to that released by the However, GPs can be used to generate sugar-1-phosphates; sugar phosphate ester cleavage (Figure 2) [4, 5]. Phosphory- in addition, coupling GP reactions with a secondary glycosy- lases have been classified into different families depending lation step provides a means of capitalizing on these robust on the sequence similarity to GTs and GHs in CAZy data- enzymes, which employ cheap and accessible substrates. base (Table 1) [4, 5]. Most of the GPs belong to GH families, [5] and they can be distinguished into retaining and inverting 1.1. Background on Glycoside Phosphorylases phosphorylases depending on whether the new glycosidic bond is formed with inversion or retention of the anomeric GPs use inorganic phosphate to breakdown glycosidic configuration with respect to the sugar-1-phosphate substrate bonds (phosphorolysis), releasing sugar-1-phosphate and (Figure 2, Table 1) [4, 5]. acceptor (Figure 1) [3-5]. The phosphorylase reaction is an equilibrium between the phosphorolysis and the reverse syn- The characterised GPs comprise enzymes that are able to thetic reaction, since the free energy released by the act on a wide range of glycosidic linkages (Table 1), with the exception for α/β-(1→6) and β-(1→1) linkages, for which no specific GPs have been characterised at the moment. *Address correspondence to this author at the Department of Biological Protein & Peptide Letters Most of the GPs known so far use D-Glc-1-phosphate as a Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK; Tel/Fax: +001-1603-450720, +001-1603-450018; donor substrate, forming disaccharides or oligosaccharides; E-mail: [email protected] far fewer GPs use D-Gal-1-phosphate [9], D-GlcNAc-1- 1875-5305/17 $58.00+.00 © 2017 Bentham Science Publishers Glycan Phosphorylases in Multi-Enzyme Synthetic Processes Protein & Peptide Letters, 2017, Vol. 24, No. 8 697 phosphate [10] or D-Man-1-phosphate [11-13] as their donor the industrial interest in GPs as a result of their cheap and substrates (Table 1) [3, 5, 14-17]. There are currently no readily available substrates, in the last decade a big effort has known phosphorylases that transfer N-acetylneuraminic acid been put into the discovery of new GP activities, as well as (Neu5Ac), L-fucose (L-Fuc), L-rhamnose (L-Rha), D- GP engineering to overcome the limitations associated with fructose (D-Fru), D-glucuronic acid (D-GlcA) or D- their strict specificity in terms of substrates or linkage [19]; glucosamine (D-GlcN) as their physiological substrates. In efforts to improve their thermostability and catalytic effi- this respect, GTs and GHs cover a broader range of carbohy- ciency with non-natural substrates have also been reported drate specificities and have been much more extensively [20, 21]. studied, characterised and modified in order to optimise their GPs have been exploited in academia and also in industry use in the synthesis of complex carbohydrates [8, 18]. for the synthesis of glycosylated compounds, high-value carbohydrates and both natural and unnatural polysaccha- -- rides. On the other hand, the phosphorolytic reaction of GPs HPO4 O HO is currently being investigated as an alternative route for R O waste recycling and biofuel production. The aim of the arti- O O O HO + HO OH cle is to provide an overview of recent developments in GP OH -- !-linkage applications, especially where they have been used in con- OPO3 I junction with other GPs (phosphorylase coupling) [8] and/or !-sugar-1-P O O HO O with other enzymes. The review will be focused on three -- HPO4 OH main classes of GPs: disaccharide phosphorylases for the "-linkage production of small molecules [5, 8, 19]; α-glucan phos- phorylases for starch synthesis and/or degradation [22, 23]; Figure 2. Reaction mechanism of glycoside phosphorylases. R, finally, cellodextrin/cellobiose phosphorylases (CDP/CBP) retaining; I, inverting. for cellulose synthesis and/or degradation [24]. All of these areas are being explored because of their potential industrial While GP synthetic reactions can be used for carbohy- application in the production of more soluble glycosylated drate/glyco-molecule synthesis, GP phosphorolysis reactions drugs, excipients, prebiotics, food additives, adhesives and can be exploited for the degradation of glyco-polymers, lead- new materials. ing to the production of useful sugar-1-phosphates. Due to Table 1. Classification of characterised glycoside phosphorylases [3]. Mechanism Family Linkage Donor Acceptor Product GT4 α,α-(1→1) α-D-Glc-1-P D-Glc Trehalose [14, 15] GT35 α-(1→4) α-D-Glc-1-P α-D-Glc-(1→4)- α-D-(Glc)n Glycogen [16]/starch [17] Retaining α,α-(1→2) α-D-Glc-1-P D-Fru Sucrose GH13 α-(1→4) α-D-Maltose-1-P α-D-Glc-(1→4)-α-D-(Glc)n α-D-Glc-(1→4)-α-D-(Glc)n+2 α,α-(1→1) β-D-Glc-1-P D-Glc/D-Glc-6-P Trehalose/trehalose-6-P α-(1→2) β-D-Glc-1-P D-Glc Kojibiose GH65 α-(1→3) β-D-Glc-1-P D-Glc Nigerose α-(1→4) β-D-Glc-1-P D-Glc Maltose β-(1→2) α-D-Glc-1-P D-Glc Sophorose β-(1→3) α-D-Glc-1-P D-Glc Laminaribiose GH94 Inverting β-(1→4) α-D-Glc-1-P D-Glc/ β-D-Glc-(1→4)-β-D-(Glc)n Cellobiose/cellodextrin β-(1→4) α-D-GlcNAc-1-P D-GlcNAc Chitobiose β-(1→3) α-D-Gal-1-P D-GalNAc/D-GlcNAc Galacto-N-biose/lacto-N-biose GH112 β-(1→4) α-D-Gal-1-P L-Rha β-D-Gal-(1→4)-L-Rha α-D-Man-1-P D-Glc β-D-Man-(1→4)-D-Glc GH130 β-(1→4) α-D-Man-1-P β-D-Man-(1→4)-β-D-(Man)n Mannan α-D-Man-1-P D-GlcNAc β-D-Man-(1→4)-D-GlcNAc 698 Protein & Peptide Letters, 2017, Vol. 24, No. 8 Pergolizzi et al. 2. DISACCHARIDE PHOSPHORYLASES by the choice of enzyme specificity and monosaccharide compositions of the product by the supplied substrates. As Disaccharide phosphorylases carry out a reversible phos- previously mentioned, relaxed acceptor preference is the key phorolytic reaction using a glycosyl phosphate and a mono- property that has been used by many research groups for the saccharide as sugar donor and acceptor, respectively. They production of rare disaccharides. Morimoto and co-workers are all members of GH and GT families that are mentioned have shown that SP from Leuconostoc mesenteroides exhib- in Table 1, except for GT35 which contains exclusively oli- its activity towards eight different ketohexose acceptors gosaccharide phosphorylases (starch and glycogen phos- while using α -D-Glc-1-phosphate as a donor, allowing the phorylases). Since the CAZy classification is based largely production of eight corresponding D-glucosyl-ketohexoses, on amino acid sequence homology [1], disaccharide phos- which are rare or absent in nature [26]. phorylases with different substrate specificities are often classified in the same family. For example, GH94 family Disaccharide phosphorylases can also be used in con- contains disaccharide phosphorylases acting on β-D-Glc- junction with other biocatalysts to expand the range of possi- (1→2)-D-Glc (sophorose), β -D-Glc-(1→4)-D-Glc (cello- ble products through a “one-pot” enzymatic approach.