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Enzyme Promiscuity of Carbohydrate Active and their applications in Pallister E, Gray CJ and Flitsch SL

School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK

Corresponding Author: Sabine Flitsch ([email protected])

Abstract The application of biocatalysis for the synthesis of glycans and glycoconjugates is a well-established and successful strategy, both for small and large scale synthesis. Compared to chemical synthesis, is has the advantage of high selectivity, but biocatalysis had been largely limited to natural glycans both in terms of reactivity and substrates. This review describes recent advances in exploiting promiscuity to expand the range of substrates and reactions that carbohydrate active enzymes (CAZymes) can catalyse. The main focus is on formation and hydrolysis of glycosidic linkages, including sugar , reactions that are central to glycobiotechnology. In addition, biocatalysts that generate sugar analogues and modify carbohydrates, such as , and acylases are reviewed. As carbohydrate active enzymes become more accessible and protein engineering strategies become faster, the application of biocatalysis in the generation of a wide range of glycoconjugates, beyond natural structures is expected to expand. Introduction Carbohydrate active enzymes (CAZymes) are responsible for the biosynthesis and metabolism of all sugars and in Nature they often have to be highly selective in a biochemically complex environment. However, as observed with other enzymes1,2, CAZymes do display ‘promiscuity’, a term that describes the capability of catalysing non-physiological secondary reactions. With the advent of high-throughput screening approaches, many examples of enzyme promiscuity have been identified and discussed in the context of evolution. For biocatalysis, promiscuity presents a good starting point to develop new reactions beyond those that are physiologically observed in Nature, 3 although the promiscuous activity is often by orders of magnitude lower than that of the native reaction.4 Given that a number of protein re- engineering tools are now available that allow for and reaction optimisation at short timescales, promiscuous activities have also become an excellent starting point for the development of new chemical processes driven by biocatalysis.5 For CAZymes, enzyme promiscuity falls into several categories of either substrate or catalytic promiscuity: Substrate promiscuity occurs when non-canonical substrates are used. As shown in Figure 1, the two partners of a glycosidic linkage are generally defined as ‘donor’ and ‘acceptor’ substrates and different enzyme classes have inherent promiscuity, depending on their physiological function. The majority of enzymes involved in synthesis and hydrolysis have strong preference for specific donor substrates, whereas the acceptor specificity can be more relaxed, and frequently be limited to requiring a specific molecular substructure motif, such as a mono or oligosaccharide, peptide or natural product. As a result, glycosidic bonds can be formed and cleaved using the same enzymes on a large diversity of glycoconjugates, both in terms of structure and size, ranging from small alcohols, thiols and amines6 to saccharides,7 peptides,8 lipids,9 DNA,10 proteins,11 arrays,12 and nanoscale glycoprotein assemblies.13

Figure 1. An overview of some frequently used reactions in biocatalysis that are catalysed by Carbohydrate Active Enzymes (CAZymes). Among the sugar-modifying enzymes, substrate specificity can be high for the terminal monosaccharides,14 but beyond the terminal motif large aglycones are tolerated, as for example in galactose , which just requires a terminal galactose.15 Remarkably, the hydrolysis of acyl-amino sugars can show high selectivity for the sugar amine, as shown for the N-acetyl glycosamine specific deacylase from a marine bacterium.16 Most studies on the catalytic promiscuity of CAZymes have focussed on the hydrolysis and synthesis of glycosidic linkages (Figure 1). In metabolism, these reactions are catalysed by very distinct classes of enzymes –Glycoside (GHs; EC 3.2.1.-) catalyse the hydrolysis and/or rearrangement of glycosidic linkages, Polysaccharide (PLs; EC 4.2.2.-) catalyse non-hydrolytic cleavage and (GTs; EC 2.4.x.y) catalyse the formation of glycosidic bonds (CAZy.com).17 Any other reaction catalysed by these classes of enzymes is generally considered as promiscuous activity. In comparison, the transamination reaction in Figure 1 is truly reversible and catalysis in either direction would be physiologically relevant. In this review, we will discuss CAZyme substrate and catalytic promiscuity by reactions as represented in Figure 1, focussing on recent developments. Sugar Phosphorylation Sugar kinases are central to metabolism, catalysing phosphorylation using ATP, as illustrated for GalK in Figure 2. Sugar 1-phosphates such as galactose-1-phosphate (Gal-P) are important metabolic intermediates for biosynthetic formation of glycosidic bonds. They have been either used directly as activated glycosides with or they are converted to sugar nucleotides (in this case UDP-Gal) which are substrates for many so called ‘Leloir’ glycosyltransferases. The interconversion of saccharides is often at the sugar nucleotide level, with UDP-glucose epimerase being a prime example that links glucosides and galactosides.18 The promiscuity of sugar kinases has been known for a while4 and has been studied in terms of evolutionary advantages to organisms. Given that glycosyl phosphates are key intermediates in biosynthesis of many diverse glycosides, an understanding of substrate tolerance is very important, first of all for the synthesis of diverse natural sugars but also for sugar analogue targets. A recent systematic study a variety of (GalKs) and N-acetyl hexosamine kinases (NahKs) demonstrated that these kinases tolerated substitutions of hydroxyl groups in the substrate well, with biocatalysts found among these classes of enzymes for a number of epimers, deoxy-sugars, deoxy-fluoro sugars and remarkably even a substrate with four fluorine substituents in the ring (Figure 2).19 A large number of analogues are also tolerated by CMP-Neu5Ac synthetase,20,21,11,16 the enzyme that catalyses the direct formation of the substrate from free sialic acid and CTP. These CMP-Neu5Ac analogues can then be applied to the synthesis of sialo- glycoconjugates, as will be discussed later (Figure 4A).

Figure 2. The formation and reactions of sugar-1-phosphates as illustrated for galactose. Kinases can be promiscuous in substrate selection and a number of synthetic analogues have been shown to be substrates for GalK and NahK with up to four fluorine ring substitutions. 19 Hydrolysis of glycosidic linkages The hydrolysis of glycosidic linkages in Nature is mostly catalysed by glycoside hydrolases (GHs; EC 3.2.1.-) which have been classified into over 165 ‘clans’ according to structure, with over 760000 sequence modules listed in the CAZyme database,22 thus providing an enormous resource. Finding desirable activity in these metagenomic data is still a great challenge, but with the access to large-scale affordable DNA synthesis, the expression and screening of libraries has become possible. An elegant example of this approach of screening glycoside hydrolases has been reported by the Withers group23 who expressed 175 GH1 enzymes and developed a high-throughput screening system that allows for both donor and acceptor screening (Figure 3A) making use of glycosynthase activity in the GH1 family. A number of sequences were found to be active against unnatural modified glucosides, such as 3-NH2, 4- NH2, 6- NH2 and 6-N3. Screening for acceptors, each glycohydrolase was found to display distinct fingerprints of aglycone specificity.

Figure 3: A. High-throughput screen applied to a synthetic gene-based library of expressing GH1 enzymes.23 B. Sialyltransferase from Photobacterium sp. can act as a 2,6 selective sialidase in the presence of CMP.28 In Nature, the hydrolysis and the formation of glycosidic bonds is each catalysed by distinct enzyme classes, and the ‘reverse’ activity is generally considered a promiscuous activity. A number of glycosyltransferases have been shown to have hydrolytic in addition to synthetic activity. A good example is the hydrolysis of sialosides, which is naturally catalysed by sialidases, of which a number have been characterised.24 The synthesis of sialosides is mediated by , which use the activated sugar nucleotides CMP-Neu-5-Ac as substrates (Figure 4B). These are widely used for glycoconjugate synthesis (as discussed later) and for some enzymes hydrolysis activity has been reported, often as an unwanted side- reaction that reduces product yields.25,26,27 However, this promiscuous activity can be used to obtain high linkage selectivity of sialoside hydrolysis, as shown in Figure 3B: here, the high 2-6 over 2-3 selectivity of a Photobacterium sp. sialyltransferase was retained in the hydrolysis direction, as demonstrated on simple trisaccharides, but also on naturally derived glycopeptides and glycoproteins. Surprisingly, all sialidases that had been reported in the literature before were either non-selective or selective for 2-3 linkages, so this ‘pseudosialidase’ provides a useful biochemical tool for the manipulation of sialoglycoconjugates.28 Formation of glycosidic linkages29,30 The enzymatic formation of glycosidic linkages, either between sugar building blocks in oligo- and polysaccharides or in glycoconjugates (Figure 1) is a very active area of research and there are a number of enzyme classes that can achieve this reaction, in particular glycosyltransferases (GTs; EC 2.4.x.y) which use activated sugar nucleotide building blocks.

B

C

Figure 4. Formation of glycosidic linkages: A: A diverse range of sialic acid analogues, in particular with substitutions at C-5 and C-9 can be generated using native aldolase and enzymatically transferred to glycoconjugates (R). B: ‘Bump-and-Hole’ engineering of GalNAc transferases. GalNAc- Ts initiate O-GalNAc glycosylation to Ser or Thr followed by downstream elongation. Double mutants (BH-GalNAc-Ts) are paired with UDP-GalNAc analogs to allow for chemical tagging using click chemistry. (reproduced with permission from ref [39]) C: Glycan remodelling of cetuximab using Endo- F3 and Endo-S and their glycosynthase mutants (reproduced with permission from ref [53]). GTs are generally highly selective for both the glycosyl donor and the acceptor (Figure 1) and catalyse the formation of glycosidic bonds with high stereo- and regio-selectivity. This led to the ‘one-linkage-one-enzyme’ hypothesis in glycan biosynthesis, which is still generally useful, but there are many exceptions to this rule. Nevertheless, in particular in vitro, GTs have shown exceptional selectivity and have been used in one-pot-enzyme cascades to generate natural glycans at preparative scale for a number of years (for a recent reviews see30,18,31). Despite the very high selectivity, the promiscuity of GTs for non-natural substrates has been exploited over recent years both for in vivo and for in vitro glycoengineering and biocatalysis. One of first and most studied sugars in this respect are sialic acids, the cell surface sugars commonly found in animal biology. N-Acetyl-neuraminic acid (Neu-5-Ac) is one of the most studied and is biosynthesized from N-acetyl mannosamine and pyruvic acid, followed by activation to CMP-Neu-5-Ac and transfer to an acceptor substrate, in animals often a terminal galactose residue. The whole biosynthetic machinery is particularly tolerant to substitutions of the 5-acetyl and the 9-hydroxyl groups (in addition to C-7 and C-8), which has led to applications in biooorthogonal labelling technologies, extensively developed and used by the Bertozzi group and others for highly specific labelling of sialic acids with azido-acetyl and other substituents at C-5. In a recent paper this bioorthogonal labelling strategy was used for the quantitative super resolution microscopy of the mammalian glycocalyx.32 Apart from azido groups, a wide range of functionalities have been incorporated into sialo glycoconjugates, as recently reviewed (Figure 4A).30 The 9-derivatisation of sialic acids has been used extensively to develop high affinity selective ligands for Siglecs, with a recent example of a 9-sulfonamide demonstrating selectivity towards Siclec8, a therapeutic target for the treatment of eosinophil and mast cell disorders.33 Both C-9 and C-5 substituted sialic acids were also used to generate ligands for siglec CD22, which allowed for the delivery of toxins in B-Lymphoma cells.34 Protein based biotherapeutics are an ever increasing field of medication used to treat a variety of diseases. Many of these biotherapeutics are glycoproteins and contain sialic acids. In vitro, sialylation has been used extensively to increase the amount of natural sialo-glycans using α2,3 and α2,6 sialyltransferases, and can also be used to introduce unnatural structures, such as a unique di-sialylgalactose motif, which was installed on alpha-1-antitrypsin.35 Given their well-studied promiscuity, sialyltransferases are ideal candidates to introduce unnatural biorthogonal chemical motifs onto antibodies for the generation of antibody-drug conjugates. This approach was used by Boons et al to introduce a reactive azide onto glycoproteins glycan via a sialyltransferase. 11 With this azide group present it was then possible with click chemistry to react the azide to chemical tags to generate a variety of antibody drug conjugates. In addition to azide incorporation, sialyltransferases have also been used to incorporate pegylated sialic acids (sialic acids with a PEG group at C5 of the sialic acid) onto a glycoprotein. PEG is known to improve half-life of certain biotherapeutics so simple enzymatic introduction of PEG via a pegylated sialic acid to a protein’s glycan is extremely useful.36 Sialyltransferases that can transfer fluorinated sialic acids to proteins are also of interest.37 This is due to fluorinated sialic acids having sialidase resistance and/or inhibition properties. However, the number of sialyltransferases that can readily transfer sialidase resistant fluorinated sialic acids is still limited. This is mainly because the fluorine containing sialic acids with the best sialidase inhibition are typically those with the fluorine at the C3 position of the sialic acid. Unfortunately, changes at the C3 position of the sialic acid are not favoured by many sialyltransferases. Finding highly active sialyltransferases for C3 fluorinated sialic acids remains an issue in the field.

The substrate promiscuity of other glycosyltransferases involved in glycan synthesis is less explored, perhaps due to the need for more complex NTP-sugar biosynthetic pathways. However, data so far suggests that many enzymes that have been described as highly selective also display (albeit lower) promiscuous activity. For example, some polypeptide GalNAc transferases (ppGalNAcTs) involved mucin biosynthesis were shown to transfer GlcNAc and Gal as well as the natural substrate to polypeptides from the corresponding UDP-sugars.8 The substrate promiscuity can be further increased by protein engineering – in two recent examples, the Bertozzi group rationally engineered a ppGalNAc-T double mutant (BH) that contained an enlarged (hole) which could tolerate UDP-GalNAc analogues with more bulky C-2 substituents (bumps) (Figure 4B).38 This ‘bump-and- hole’ approach was used on several ppGalNAcT isoforms and could be applied subsequently to the chemical precision glyco-mutagenesis in living cells, looking at individual isoforms of ppGalNAcTs.39 Bacterial GTs have also been found that display considerable promiscuity especially for structures with small perturbations from the natural substrate. For example GtfE, a responsible for the first glycosylation step in the biosynthesis of vancomycin, shows remarkably lax substrate tolerance accepting glycosyl donors that are hexose isomers (Man, Gal, idose, talose etc.) of the natural substrate (Glc), other non-hexose natural sugars (GlcNAc, Xyl, etc.) and unnatural substrates (3-deoxy-3-azido-D-glucose, 6-deoxy-6-azido-D-glucose, 6-deoxy-6-thio- D-glucose etc.) leading to the concept of ‘glycorandomisation’.36 Very recently, the permissive GT OleD Loki was shown to convert a diverse set of >15 (HDAC) inhibitors (HDACis) into their corresponding hydroxamate glycosyl esters.40 One of the major challenges in the field is how protein sequence relates to substrate specificity, an issue that is being started to address as more structure-function relationships are being recorded based on emerging biochemical and structural datasets. 41,42 Glycan phosphorylases43 have advantage over the above ‘Leloir’ GTs in that they only require donor activation as the glycosyl phosphate and can be much more relaxed for acceptor specificity. This promiscuous activity was recently used to construct a number of new-to-nature analogues of human milk oligosaccharides.44 Phosphorylases can also use disaccharides such as the cheaply available sucrose, which was recently demonstrated in a three-enzyme cascade that produced short- chain cellodextrins.45 from B.megaterium was recently engineered to produce fructo-oligosaccharides from sucrose with different degrees of polymerization, depending on active site mutations.46 A further class of enzymes that have been used for glycosidic bond formation are glycosylhydrolases (GHs) ‘in reverse’ using various strategies to favour the promiscuous synthetic over natural hydrolytic activity, or using them as transglycosylating catalyst, ie replacing water with alcohols and other nucleophiles.23,27 GH mediated synthesis can generally be accomplished by two means: shifting the equilibrium by using high concentrations of sugars or low water systems; or application of a highly reactive sugar donor (e.g. glycosyl fluorides) in combination with active site mutagenesis to suppress hydrolysis (glycosynthases) (Figure 3A).23 Using GHs and glycosynthases is a very popular field of research47,48,49 because of the number of metagenomics data available on these enzymes, simple donor requirements, wide acceptor promiscuities and general robustness of GHs. A comprehensive coverage of all recent papers goes beyond this paper and the reader is referred to recent reviews.50,51 As well as exo-GHs and their mutants, endo-glycohydrolases offer an exciting opportunity for enzymatic synthesis, especially homogenous “remodeling” of N-glycoproteins exploiting endo-β-N-acetylglucosaminidases (EC 3.2.1.96).52 Several natural endo-β-N-acetylglucosaminidases have been characterized, that catalyze hydrolysis between the N,N’-diacetylchitobiose core within N-glycans leaving a truncated oligosaccharide with a terminal oxazoline and the core GlcNAc linked to the polypeptide chain through asparagine as in 1 (Figure 4C). This reaction has been shown to be reversible, providing the opportunity to couple complex sugars back onto the polypeptide, when activated as an anomeric oxazoline such as 2. Protein engineering of the ‘ENGases’ has generated mutants with high transfer activity, which has allowed for the glycoengineering of peptides and biopharmaceuticals. In a recent example, glycan remodeling of cetuximab was demonstrated by using wildtype Endo-F3 and Endo-S for cleavage to 1 and glycosynthase mutants Endo-F3 D165A and Endo-S D233Q for generation of heavily glycosylated homogeneous antibodies 3 and 4 (Figure 4C). 53 The glycoengineered cetuximab was shown to have significantly enhanced antibody-dependent cell- mediated cytotoxicity (ADCC) activity. Further examples of this glycoengineering are the one-pot remodeling of rituximab54 and of erythropoietin55 demonstrating wide applicability to important biotherapeutics. Initially, this Endo based technology was limited by the need for oxazolines at the reducing end, which often required total synthesis of the glycosyl donor. A significant breakthrough has been the use of DMC (2-chloro-1,3-dimethylimidazolium chloride) and related reagents for converting unprotected oligosaccharides (that can be isolated from natural sources) containing reducing 2-acetamido sugars into the corresponding oxazoline in water.56,57 Sugar modifications Enzymatic strategies for sugar functionalisations both at monosaccharide and glycoconjugate level are of great interest because chemical strategies can be unselective and limited. A very useful biocatalytic reaction is the oxidation of specific primary hydroxyl groups, such as catalysed by (Figure 5A). This copper-dependent enzyme is highly selective for terminal galactose residues, even in the context of complex cellular environments. The resulting aldehyde can be considered as a biorthogonal group and be reacted selectively with a number of chemical reagents and labels. Considerable efforts have been made to evolve galactose oxidases into catalysts with more promiscuous activities, including terminal glucose, 58 mannose59 and N-glycolyl-neuraminic acid,60 retaining its broad tolerance towards large glycoconjugates, including DNA-encoded libraries.10

Any carbonyl-groups in sugars can potentially be converted to amino-groups using transaminases in tandem with oxido- to generate novel valuable amine functionalised building blocks for biomaterials applications.61 In a recent report the direct aminations free sugars such as 2-deoxy ribose, arabinose, 2-deoxy galactose, lyxose and rhamnose to amino-alcohols using commercially available ATA256 were demonstrated on preparative scale (Figure 5B).62 As discussed in an earlier section on sialic acids, amino groups are frequently acetylated and provide an opportunity to introduce biooorthogonal functionality into sugars and glycoconjugates. Any unnatural substituent is often introduced using chemical multistep processes, but recently a highly glucosamine-specific acylase has been characterized from Cyclobacterium marinum 63,16 which allowed for the acylation with a range of carboxylic acids, resulting in an enzyme cascade starting from glucosamine directly to sialosides (Figure 5B).

Figure 5. Modification of carbohydrates and glycoconjugates using enzymes. A: galactose oxidase catalyses the selective C-6 oxidation of terminal galactosides; the F2 mutant, obtained through , has broader promiscuous activity, including the oxidation of terminal Neu-5-glycolyl residues in glycoproteins. B: Enzymatic transformations of amino-sugars using transaminases and acylases Conclusions and Outlook In the context of a natural cellular environment, CAZymes have evolved to be highly substrate and reaction selective, properties that make them valuable for in vitro biocatalysis as well. The advantage of biocatalysis for the synthesis and manipulation of sugars is that they are easy to use by bioscientists and can often been performed on very complex biomolecules, as demonstrated in this review. Although the natural reaction toolbox is limited, there are now increasing numbers of examples where CAZymes can show low promiscuous activity against other non- natural substrates, and these have been successfully exploited for in vitro synthesis. A deep understanding of enzyme mechanisms has also led to very elegant redesign of function, as seen for the glycosynthases. There are now many strategies available to scientists looking for desirable promiscuous CAZyme activities, including information of metagenomics sequences compiled in databases such as CAZy.com,64 large scale expression studies of homologues, high throughput screening strategies and protein engineering. These promiscuous activities have become excellent starting points for further catalyst development. Acknowledgements This work was supported by ERC (788231-ProgrES-ERC-2017-ADG) EU (CarboMet 737395) BBSRC (IBCarb BB/L013762/1; BB/M027791/1; BB/M02903411; BB/M028836/1) to C.J.G and S.L.F.

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