International Journal of Molecular Sciences Review Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach Luuk Mestrom 1, Marta Przypis 2,3 , Daria Kowalczykiewicz 2,3, André Pollender 4 , Antje Kumpf 4,5, Stefan R. Marsden 1, Isabel Bento 6, Andrzej B. Jarz˛ebski 7, Katarzyna Szyma ´nska 8, Arkadiusz Chru´sciel 9, Dirk Tischler 4,5 , Rob Schoevaart 10, Ulf Hanefeld 1 and Peter-Leon Hagedoorn 1,* 1 Department of Biotechnology, Delft University of Technology, Section Biocatalysis, Van der Maasweg 9, 2629 HZ Delft, The Netherlands; [email protected] (L.M.); [email protected] (S.R.M.); [email protected] (U.H.) 2 Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, B. Krzywoustego 4, 44-100 Gliwice, Poland; [email protected] (M.P.); [email protected] (D.K.) 3 Biotechnology Center, Silesian University of Technology, B. Krzywoustego 8, 44-100 Gliwice, Poland 4 Environmental Microbiology, Institute of Biosciences, TU Bergakademie Freiberg, Leipziger Str. 29, 09599 Freiberg, Germany; [email protected] (A.P.); [email protected] (A.K.); [email protected] (D.T.) 5 Microbial Biotechnology, Faculty of Biology & Biotechnology, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum, Germany 6 EMBL Hamburg, Notkestraβe 85, 22607 Hamburg, Germany; [email protected] 7 Institute of Chemical Engineering, Polish Academy of Sciences, Bałtycka 5, 44-100 Gliwice, Poland; [email protected] 8 Department of Chemical and Process Engineering, Silesian University of Technology, Ks. M. Strzody 7, 44-100 Gliwice Poland.; [email protected] 9 MEXEO Wiesław Hreczuch, ul. Energetyków 9, 47-225 K˛edzierzyn-Ko´zle,Poland; [email protected] 10 ChiralVision, J.H. Oortweg 21, 2333 CH Leiden, The Netherlands; [email protected] * Correspondence: [email protected]; Tel.: +31-15-278-2334 Received: 2 October 2019; Accepted: 18 October 2019; Published: 23 October 2019 Abstract: 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. Keywords: glycosyltransferase; applied biocatalysis; enzyme cascades; chemoenzymatic synthesis; sugar chemistry; carbohydrate; Leloir; nucleotide 1. Introduction Enzymes were already used for the conversion of glycosides even before all stereochemical details of the known carbohydrates were assigned [1,2]. In 1837, a crude formulation of almonds containing Int. J. Mol. Sci. 2019, 20, 5263; doi:10.3390/ijms20215263 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 2 of 35 1. Introduction Int. J. Mol.Enzymes Sci. 2019 were, 20, 5263 already used for the conversion of glycosides even before all stereochemical2 of 36 details of the known carbohydrates were assigned [1,2]. In 1837, a crude formulation of almonds containing hydroxynitrile lyases catalyzed the enzymatic hydrolysis of the glycoside amygdalin [3]. hydroxynitrileMoving almost lyases two centuries catalyzed forward, the enzymatic the largest hydrolysis volumetric of the biocatalytic glycoside amygdalinindustrial process [3]. Moving is the almostapplication two centuries of glucose forward, isomerase the largest for the volumetric production biocatalytic of high industrial fructose processsyrup for is the food application and drink of glucoseapplications, isomerase producing for the productionfructose from of highglucose fructose at 10 syrup7 tons for per food year and [4]. drink The applications,secret of the producingsuccess of fructoseenzymes from in the glucose production at 107 tons or treatment per year [ 4of]. Thecarboh secretydrates of the and success glycosides of enzymes is their in the exquisite production stereo- or treatmentand regioselectivity. of carbohydrates The excellent and glycosides selectivity is theirof enzy exquisitemes is required stereo- and due regioselectivity. to the diversity The of structural excellent selectivityfeatures of of carbohydrates enzymes is required [5], comprising due to the D- diversity and L-epimers, of structural ring featuressize, anomeric of carbohydrates configuration, [5], comprisinglinkages, branching,d- and l-epimers, and oxidation ring size,state(s). anomeric Since drug configuration, targets often linkages, exhibit branching, specificity andfor all oxidation of these state(s).structural Since features, drug targets the production often exhibit process specificity should for allnot of thesecontain structural any side-products features, the productionto prevent processundesired should side-effects not contain [6]. any side-products to prevent undesired side-effects [6]. TheThe challengechallenge inin thethe synthesissynthesis ofof carbohydratescarbohydrates isis theirtheir widewide varietyvariety ofof functionalitiesfunctionalities andand stereochemistrystereochemistry (Figure (Figure1 ).1). (Poly)hydroxyaldehydes (Poly)hydroxyaldehydes containing containing aterminal a terminal aldehyde aldehyde are are referred referred to as to aldosesas aldoses and and (poly)hydroxyketones (poly)hydroxyketones are definedare defined as ketoses. as ketoses. In aqueous In aqueous solutions, solutions, monosaccharides monosaccharides form equilibriumform equilibrium mixtures mixtures of linear of open-chain linear open-chain and ring-closed and ring-closed 5- or 6-membered 5- or 6- membered furanoses orfuranoses pyranoses, or respectively.pyranoses, respectively. For aldoses, For the aldoses, asymmetric the asymmetric ring forms at ring C-1. forms For ketoses, at C-1. For it closes ketoses, at C-2 it closes as an axialat C-2 ( αas) oran equatorialaxial (α) (orβ) hemiacetalequatorial ( orβ) hemiketal,hemiacetal respectively or hemiketal, (commonly respectively defined (commonly as the anomeric defined center). as the Aanomeric glycosidic center). linkage A is glycosidic a covalent Olinkage-, S-, Nis-, ora Ccovalent-bond connecting O-, S-, N a-, monosaccharide or C-bond connecting to another a residuemonosaccharide resulting to in another a glycoside, residue while resulting glucoside in a isglycoside, specific forwhile a glucose glucoside moiety. is specific The equatorialfor a glucose or axialmoiety. position The equatorial of the glycosidic or axial bond position is referred of the glycosidic to as α- (axial) bond or isβ referred-linkage to (equatorial). as α- (axial) The or β number-linkage of(equatorial). carbohydrates The linkednumber via of glycosidic carbohydrates bonds linked can be via subdivided glycosidic into bonds oligosaccharides can be subdivided with two into to tenoligosaccharides linked carbohydrates, with two while to ten polysaccharides linked carbohydrates, (glycans) while contain polysaccharides more than ten glycosidic(glycans) contain bonds. Amore glycan than either ten glycosidic contains multiplebonds. A di glycanfferent either monosaccharides contains multiple or more different than ten monosaccharides glycosidic bonds. or Amore glycoconjugate than ten glycosidic contains bonds. at least A glycoconjugate one or more monosaccharides contains at least one or oligosaccharidesor more monosaccharides covalently or attachedoligosaccharides to a non-carbohydrate covalently attached moiety to (aglycon). a non-carbohydrate If an oligosaccharide moiety (aglycon). contains If an an aldose oligosaccharide or ketose thatcontains is in equilibriuman aldose or with ketose its open-chainthat is in equilibrium form, the aldehydewith its open-chain or ketone can form, be oxidizedthe aldehyde with or chemical ketone reagentscan be oxidized (e.g., with with the chemical Benedict reagents reagent). (e.g., This iswith referred the Benedict to as the reagent). reducing This end is in referred oligosaccharides. to as the Ifreducing there is noend possibility in oligosaccharides. for the sugar If there to form is no the possibility open chain-form, for the sugar then to this form is called the open a non-reducing chain-form, end.then this Non-reducing is called a sugars non-reducing are found end. in glycoconjugatesNon-reducing sugars (i.e. nucleotides)are found in and glycoconjugates oligosaccharides (i.e. (i.e.,nucleotides) raffinose). and oligosaccharides (i.e., raffinose). Figure 1. The nomenclature of glycosides and oligosaccharides. Figure 1. The nomenclature of glycosides and oligosaccharides. Glycosyltransferases (GTs) catalyze the transfer of a carbohydrate acceptor from an activated sugar nucleotide donor with high selectivity and yield, enabling the stereo- and regioselective extension and branching of large glycans and glycoconjugates (Scheme1). Upon formation