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98 nature structural biology • volume 8 number 2 • february 2001

Sweet secrets of synthesisGideon J. Davies

The crystal structure of a ‘retaining’ glycosyltransferase provides the first view of α-glycosidic bond synthesisusing activated nucleotide sugars.

These are exciting times for glycobiology.Simply in terms of quantity, the formationof glycosides such as di-, oligo- and poly-saccharides and various glycoconjugates,is the most important biological reactionon earth. Yet despite their profoundimportance, the enzymes involved in thisprocess — nucleotide-sugar dependentglycosyltransferases — have until recentlyremained one of the great uncharted areasof structural and mechanistic enzymolo-gy. On page 166 of this issue of NatureStructural Biology, however, Persson andcolleagues1 reveal the structure of anenzyme that makes α-glycosidic linkagesusing activated α-linked nucleotide-sugardonors — a so-called ‘retaining’ glycosyl-transferase. The alpha-1,4-galactosyl-transferase C (lgtC) from Neisseriameningitidis provides our first view ofhow nature synthesizes glycosidic linkageswith retention of anomeric configuration.The structure determination and analysisinvolved the harnessing of specificallysynthesized UDP-sugar and oligosaccha-ride analogs, site-directed mutagenesisand kinetics. This work has importantramifications for researchers in glycobiol-ogy, especially those in the quest for newtherapeutic agents.

Over the past decade, our understand-ing of the structures and mechanismsinvolved in the hydrolysis of glycosides hasblossomed (for reviews see refs 2,3). Yetknowledge of the enzymes and mecha-nisms involved in the synthesis of di-,oligo- and polysaccharides has remainedelusive. This reflects both the difficulty inexpressing these enzymes, which are fre-quently membrane-bound, and the prob-lems in characterizing enzymes whosesubstrates are, potentially, enormouslycomplex and extremely challenging tosynthesize (for example, a hexasaccharidehas 1012 possible isomers).

Sequences and families ofglycosyltransferasesAt the sequence level there is now a largenumber of open reading frames that cor-respond to glycosyltransferases. Thesequence family classification that forms

the foundation of research into glycosidehydrolases has now been extended to theactivated-sugar dependent glycosyltrans-ferases and currently reveals 49 potentialfamilies (available at the carbohydrate-active enzymes server4, CAZy,http://afmb.cnrs-mrs.fr/∼ pedro/CAZY/).Assigning a function to these ORFsremains tricky. A handful of amino acidsubstitutions may change the specificity ofa transferase, and some enzymes evenchange their bond specificity dependingon the nature of the substrates involved.Furthermore, like many of the enzymesacting on carbohydrates, glycosyltrans-ferases are frequently multi-modular, inthat they are often composed of indepen-dent domains within the same peptidechain. This poses additional problems forcorrect genomic annotation5. The bigchallenge for structural glycobiologists,therefore, is to dissect the structure andfunction of the enzymes in these 49 fami-lies. Put more simply, how does naturemake glycosidic bonds? The structurereported by Persson and colleagues1 goes along way toward answering some of theunresolved issues.

The enzymatic synthesis ofglycosidesThe enzymatic synthesis of glycosidicbonds in nature involves several different

strategies. Glycoside hydrolases, whichmay normally be considered degradativeenzymes, may, under favorable condi-tions, act ‘in reverse’ to synthesize oligo-and polysaccharides (for example seeref. 6). This strategy is limited becausemost reactions take place in aqueous solu-tion and hydrolytic (degradative) process-es are usually favored. The vast majority ofglycosidic bonds in nature are, therefore,instead synthesized using ‘activated’ sugarprecursors. Such compounds possessgood leaving-groups which drive the reac-tion in favor of synthesis. Typically theleaving group is a lipid-phosphate sugaror a nucleotide-sugar, such as the UDP-galactose utilized by lgtC.

As with hydrolytic enzymes, we mayconsider two general classes of glycosyl-transferases based upon whether theyinvert or retain the anomeric configura-tion. The cyclized or ‘ring’ forms of sugarspossess what is termed an ‘anomeric’ car-bon. In the linear form of a sugar this atomis achiral, but upon cyclization it acquireschirality and may display one of twostereochemistries termed α or β. Enzymesthat perform nucleophilic substitution atthis special anomeric center, including gly-coside hydrolases and glycosyltransferases,therefore form two general classes basedupon whether they invert or retain thestereochemistry at this center.

Fig. 1 Reaction mechanism for an inverting NDP-sugar glycosyltransferase. The reaction involves asingle displacement in which an activated sugar donor is attacked by the acceptor leading toinversion of anomeric configuration. A Brønsted base aids nucleophilic attack by deprotonatingthe acceptor species and often a divalent metal ion assists leaving group departure. The recentrabbit N-acetylglucosamine transferase11 and human glucuronyltransferase12 structures providefine views of sugar donor and acceptor species on inverting enzymes, respectively.

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nature structural biology • volume 8 number 2 • february 2001 99

In the case of nucleotide-sugar depen-dent glycosyltranferases the nucleotide-sugar donor is invariably an α-linkedspecies. Inversion therefore gives rise to aβ-linked product (that is, one whoseanomeric configuration is inverted rela-tive to the substrate) while retention ofanomeric configuration gives rise to an α-linked product whose stereochemistryis identical to that of the starting sugardonor. At the mechanistic level it is theselatter, retaining, enzymes that have gener-ated the most intrigue. The inversionmechanism may easily be envisaged (Fig.1) but the intimate secrets of retainingenzymes are less easily revealed.Furthermore, while the last few monthshave witnessed the determination of anumber of structures of enzymes involvedin glycosidic bond formation7–12 all thesehave been of the conceptually easierinverting category.

The breakthrough made by the Perssonet al.1 is that they have determined the firststructure of lgtC, a configuration-retain-ing glycosyltransferase involved in thesynthesis of the major glycolipid on thecell surface of N. meningitidis. LgtC is amembrane-associated protein that cat-alyzes the addition of galactose, from

UDP-galactose, to the terminal lactosemoiety on the lipopolysaccharide. Thestructure was determined in complex withboth nucleotide-sugar donor and sugaracceptor analogs, which are themselvesno mean feat of organic synthesis.

As with the inverting transferase struc-tures (for review see ref. 13) the lgtCstructure forms an α/β protein with an N-terminal nucleotide-binding fold inwhich the inert UDP-sugar analog bindsin a shallow cleft. The C-terminal regionforms the binding site for the acceptorspecies, which has been visualizedthrough the use of an acceptor analog, 4-deoxylactose. Together, the positions ofthe UDP-2F sugar and the acceptor analogbegin to shed light upon the catalyticmechanism of these enzymes.

The most important question concern-ing retaining glycosyltransferases is todetermine what chemical mechanism isutilized for glycosidic bond formation.The mechanism for glycosidic bondhydrolysis, by glycoside hydrolases, withnet retention of anomeric configuration isnow very well-understood. Hydrolysis bythis class of enzymes occurs in a double-displacement reaction via the formationand subsequent hydrolysis of a covalent

intermediate species. Yet while retainingglycoside hydrolases are well-character-ized, the analogous synthetic reaction car-ried out by the activated-sugar dependentglycosyltransferases remains unclear. Theanalogy with glycosidases suggests a cova-lent intermediate, but such a proposallacks any experimental evidence. In such adouble-displacement mechanism (Fig. 2a)one would expect a nucleophile to lie closeto the correct (β) face of the UDP-sugardonor. In the quest for a solution Perssonand colleagues1 mutated and analyzed allsuch potential catalytic nucleophiles in thevicinity of the UDP-sugar, yet no mutationgenerated an enzyme with activity reducedenough to suggest deletion of a catalytical-ly essential nucleophile. The authors thenconsidered a novel mechanism in which asugar hydroxyl from the lactosyl acceptoracts as a nucleophile in a two-step reaction;again the results with specifically synthe-sized sugar analogs were inconclusive. Afurther possibility proposed by the authorsis that the enzyme utilizes an extremelyunusual SNi ‘internal return’ mechanism inwhich nucleophilic attack by the acceptorand leaving-group departure occur simul-taneously on the same face of the sugar(Fig. 2b). While such a mechanism is not

Fig. 2 Possible mechanisms for a retaining NDP-sugar glycosyltransferase. a, A reaction thatoccurs, by analogy with retaining glycosidehydrolases, via a covalent intermediate. Perssonand colleagues1 consider both enzymatic and acceptor-derived intermediates. b, An unusualSNi-like ‘internal return’ transition state inwhich both departing and attacking groupsexist on the same side of the sugar ring.

a

b

a b

Fig. 3 The active center of lgtC is reminiscent ofglycogen and maltodextrin phosphorylases.Comparison of a, a pseudo-substrate complex oflgtC with a UDP 2-fluorogalactoside and 4-deoxylac-tose (PDB code 1GA8) and b, a pseudo-product com-plex of maltodextrin phosphorylase with thepyridoxal phosphate cofactor, phosphate plus a thio-oligosaccharide (1QM5)21. The unusual folded-backconformation of the α-linked UDP-Gal is similar tothe glucose and phosphate positions in maltodextrinphosphorylase. In both cases a carbonyl group sits∼ 3.3Å ‘above’ the β-face of the anomeric carbon. InlgtC this carbonyl is provided by Gln 189 and in mal-todextrin phosphorylase it is provided by the mainchain carbonyl of His 345.

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100 nature structural biology • volume 8 number 2 • february 2001

without chemical precedent, it would beextremely unusual. It has, however, alsopreviously been proposed for glycogenphosphorylase18, an enzyme that bears agreat deal of mechanistic similarity to lgtC.

Similarities with glycogenphosphorylaseIndeed, one of the extremely revealing fea-tures of the lgtC structure is this similaritywith glycogen phosphorylase. Glycogenphosphorylase itself may also be consid-ered a retaining glycosyltransferase (albeitnot one that uses nucleotide-sugars), sinceit utilizes glucose-1-phosphate as the acti-vated sugar donor to synthesize α-1,4 gly-cosidic bonds in a reversible manner. Aswith the retaining nucleotide-sugardependent transferases, its mechanismremains unclear. A mechanism has beenproposed based upon crystal structuresthat involves a long-lived oxocarbenium-ion intermediate, but this mechanism isnot widely believed by chemists18,19. Aswith lgtC, a covalent reaction intermedi-ate would seem most likely but again nonehas ever been identified. It has even beenproposed that a main chain carboxylatefunctions as a nucleophile or that theenzyme uses the unusual SNi mechanismdescribed above.

There are great parallels between theway the UDP-sugar binds to lgtC, in anunusual folded-back conformation, andthe binding of the pyridoxal 5′-phosphate(PLP) cofactor, glucose and phosphate inglycogen phosphorylase which again hints

that these two enzymes may share similarreaction mechanisms (Fig. 3). The PLPgroup of phosphorylase mimics the UMPmoiety of the UDP-Gal in lgtC while theGlc-1-phosphate of phosphorylase liessimilarly positioned to the galactosyl-phosphate moiety of the UDP-sugar inlgtC. Furthermore, the main chain car-bonyl postulated as a nucleophile forphosphorylase (from His 345 in the caseof E. coli maltodextrin phosphorylase),overlays the side chain carbonyl of Gln 189 in lgtC. While both are positionedclose to the β-face of the sugar donor,mutation of Gln 189 in lgtC suggests thatit does not play the role of an enzymaticnucleophile. The true catalytic mecha-nism of both glycogen phosphorylase andlgtC therefore remains a mystery.

Glycosyltransferases are central to allsynthetic processes involving carbohy-drates. They are important drug targets inthe fight against cancer as well as bacterial,viral and fungal infections because theyoften perform organism and cell-specificprocesses. They also offer enormouspotential for the chemoenzymatic synthe-sis of oligosaccharide-based therapeuticagents20. Knowledge of their three-dimen-sional structures and reaction mechanismsis essential for the exploitation of theseenzymes as catalysts and drug targets. ThelgtC structure provides our first glimpse ofa retaining glycosyltransferase and offerspreliminary illumination of this complexarea. Yet, while the lgtC structure, skillfullyanalyzed with both donor and substrate

analogs, gives us the first view of the syn-thetic apparatus for α-linked sugars andreveals the location of appropriate chemi-cal functions, the exact mechanism ofcatalysis remains unclear. Nature has notrevealed all her sweet secrets just yet.

Gideon Davies is at the Structural BiologyLaboratory, Department of Chemistry,University of York, Heslington, York YO105DD, UK. email: davies@ysbl.york.ac.uk

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10. Ha, S. et al. Protein Sci. 9, 1045–1052 (2000).11. Ünligil, U.M. et al. EMBO J. 19, 5269–5280 (2000).12. Pedersen, L.C. et al. J. Biol. Chem. 275,

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406–408 (1999).18. Sinnott, M.L. Chem. Rev. 90, 1171–1202 (1990).19. Davies, G.J. et al. In Comprehensive biological

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