The Cyclization Mechanism of Cyclodextrin Glycosyltransferase (CGTase) as Revealed by a γ-Cyclodextrin-CGTase Complex at 1.8-Å Resolution

,

An especially interesting and unique family 13 enzyme is cyclodextrin glycosyltransferase (CGTase). 1CGTase forms circular ␣(134)-linked oligoglucosides called cyclodextrins from linear ␣(134)-linked glucans via an intramolecular transglycosylation reaction known as cyclization (see Fig. 1).In addition, CGTase can perform hydrolysis (very poorly) and disproportionation reactions by using water or a free oligosaccharide as respective acceptors.Its ability to perform cyclization has led to the application of CGTases in the industrial production of cyclodextrins (4).
CGTases are typically 75-kDa proteins consisting of 5 domains (A-E) (10,11).Domains A and B constitute the catalytic domains, whereas C and E are specialized in binding to raw starch granules (11,12).Domain D has no known function.The active site of Bacillus circulans strain 251 CGTase was shown to comprise at least nine sugar binding subsites (13), labeled Ϫ7 to ϩ2.Bond cleavage occurs between subsites Ϫ1 and ϩ1 (see Fig. 1), resulting in an intermediate that is covalently linked to Asp-229 at subsite Ϫ1 (7).During cyclization, CGTase circularizes the linear chain of this intermediate (7) such that the nonreducing end glucose residue moves from subsite Ϫ7 into subsite ϩ1.There it is used as the acceptor sugar and becomes covalently linked to the C1 atom of the sugar at subsite Ϫ1 (see Fig. 1).We denote this sugar transfer step circularization to distinguish it as a subprocess of cyclization.Astonishingly, circularization requires a 23 Å-movement of the linear chain end (13).Kinetic data suggest that this is the rate-limiting step in the cyclization reaction. 2 The mechanism of cyclization has been extensively studied to gain insight into the factors that determine the substrate specificity of the enzyme and the reaction type specificity and to improve CGTase for industrial applications.Site-directed mutagenesis studies covering almost all sugar binding subsites show that specific residues at subsites ϩ2, Ϫ3, and Ϫ6 contribute to cyclization (reviewed in Table I, see "Discussion").Of particular importance is the centrally located residue Tyr-195, as was demonstrated in at least 6 different CGTases (Table I).To understand how these residues confer cyclization activity, structural information about the individual stages of the reaction cycle is essential.
Crystallographic investigations have already revealed how a maltononaose substrate and a covalent maltotriosyl intermediate bind in the active site of CGTase (7,13).(Fig. 1).However, it is unknown how CGTase binds a cyclodextrin product.A structure of a CGTase-␤-cyclodextrin derivative complex has been determined, but this shows a cyclodextrin that has already partly departed from the catalytic site (14).Here we present the 1.8-Å structure of a ␥-cyclodextrin that is competently bound in the active site of B. circulans strain 251 E257Q/ D229N CGTase.Our data provide for the first time detailed insights in the mechanisms that are employed by CGTase to perform the cyclization reaction.

EXPERIMENTAL PROCEDURES
Soaking Procedure-Crystals of B. circulans strain 251 CGTase were grown from 60% (v/v) 2-methyl-2,4-pentanediol (MPD), 100 mM HEPES, pH 7.55, and 5.0% (w/v) maltose (11).In the past, various x-ray structures of CGTase-oligosaccharide complexes have been obtained after soaking crystals in a mother liquor where the maltose had been replaced by another sugar or inhibitor (13,15,16).However, when cyclodextrins are used for soaking, they are digested to small linear oligosaccharides (15).To prevent this, we used crystals of the marginally active E257Q/D229N CGTase (15) equilibrated at pH 10.3.One of these crystals was soaked for 30 min in a solution containing 60% MPD and 100 mM CAPS buffer at pH 10.3 saturated with ␥-cyclodextrin.Thereafter it was frozen under a cold nitrogen stream (120 K) for data collection using the soaking solution as cryo-protectant.
Data Collection and Refinement-Data to 1.78 Å were collected at beamline X11 of the EMBL outstation at DESY, Hamburg.The data were processed and refined using standard crystallographic techniques (17) involving DENZO (18), O (19), TNT (20), and ARP (21).Normal and OMIT (22) electron density maps were calculated with the BIOMOL package, applying the SIGMAA procedure (23).As a starting model, we used the 2.2-Å structure of unliganded wild type CGTase at pH 7.55 and 120 K (15).From the initial electron density map it was obvious that three ␥-cyclodextrins were bound, one in the active site and two at the raw starch binding sites in the E-domain (MBS1 and MBS2 (11)).At MBS3 in the C-domain a maltose was observed, as in other CGTase structures (11).The ␥-cyclodextrins were built into their electron density and refined using the stereochemistry from their crystal structure (24).
Halfway through refinement we observed density for a MPD molecule near residues Pro-379 and Arg-377, which was included in the model using stereochemical parameters from the hetero compounds data base (G.Kleywegt, Uppsala University, Uppsala, Sweden).One water molecule refined to a suspiciously low B-factor of 2.2 Å 2 .It had an F o Ϫ F c OMIT electron density peak of 17 times the standard deviation and was surrounded by the atoms Ala-315 O at 2.3 Å and Asp-577 O␦1 (in a new orientation) at 2.7 Å as well as 3 water molecules at a 2.4-Å distance.The peak height and ligation suggested the presence of a third Ca 2ϩ binding site in addition to the two Ca 2ϩ sites observed earlier (11).It is located far from the active site, at the interface of the A and D domains.Inspection of other data sets revealed a similar peak in a 1.5-Å-unliganded CGTase structure at pH 10.3 3 but not in any other structure determined at a lower pH.Therefore, most likely this third Ca 2ϩ site is an artifact of the high pH used, with little relevance for the activity of CGTase at optimal pH (pH 5.5).
In the final stage of the refinement the orientations of His, Gln, and Asn residues were determined by optimization of their hydrogen-bonding networks (25).In addition, the Asn and Gln orientations were verified by the difference in atomic B-factors of their O␦/⑀ and N␦/⑀ atoms (with the program ACT (26)).The stereochemistry of all amino acids was verified with PROCHECK (27) and WHATCHECK (25).The final electron density for the ␥-cyclodextrin bound in the active site is shown in Fig. 2. Data and refinement statistics are given in Table II.
Verification of the Results-To exclude artifacts arising from the pH, mutations, or temperature, we first compared the structures of unliganded E257Q/D229N CGTase at 120 K, pH 10.3, 3 with unliganded wild type CGTase at 120 K, pH 7.6 (15), and 293 K, pH 7.6 (11).No significant differences were observed.Second, we restricted functional comparisons to CGTase structures that were determined under identi- a Residues delimiting the subsite surface with their side chains.
b Identification based on a superposition of 3-dimensional structures.Dashes denote the absence of the corresponding residues in ␣-amylases.
c On the basis of 20 CGTase sequences which were downloaded using Entrez.Boldface indicates that a residue is conserved in CGTase but not in ␣-amylases.Amino acid variations at a position are separated by a slash, with the most frequently occurring type given first, with a dash denoting a deletion.If a variation comprises more than three amino acid types, two dots are given in fourth position.
d The residue function is derived from the relatively most decreased type of activity after site-directed mutagenesis, and specificity refers to the cyclodextrin size ratio produced.
e Function based on unpublished results.f Mammalian ␣-amylases have a conserved Ala at position 230; therefore Ala 230 is not indicated as typical for CGTase.
The Cyclization Mechanism of CGTase cal experimental conditions, which warrants that all observed differences are ligand-induced.

RESULTS
Binding of the ␥-Cyclodextrin in the Catalytic Site of CG-Tase-In the CGTase-cyclodextrin complex, two ␥-cyclodextrin molecules are bound at the maltose binding sites (MBS) MBS1 and MBS2 in the E domain, and one is bound in the active site.The electron density clearly shows that the cyclic symmetry of the latter CD is spectacularly deformed (Figs. 2 and 3) by the catalytic site architecture that has forced the ϩ1 glucose out of the ring structure.As a result, the torsion angles of the scissile bond have acquired a characteristic twist (16).This brings the glucoses at subsites Ϫ1 and ϩ1 close to the catalytic residues and makes them superimpose well with other CGTase and ␣-amylase ligand complexes (16,28,29).This assures us that the ␥-cyclodextrin is bound in a catalytically competent fashion, in contrast to the earlier CGTase-␤-cyclodextrin complex (14).
Comparison of ␥-Cyclodextrin and Maltononaose Substrate Binding Modes-To gain insight into the cyclization mechanism, the binding mode of ␥-cyclodextrin was compared with that of a linear maltononaose substrate bound from subsites Ϫ7 to ϩ2 (7).Both structures were determined under identical experimental conditions.A superposition is shown in Fig. 3.At subsites Ϫ2, Ϫ1, and ϩ1, both ligands are bound in a nearly identical fashion.Both show the "twist" discussed above and also distortion of the glucose ring at subsite Ϫ1 toward transition state planarity (7).In contrast, at subsites ϩ2 and Ϫ3, ␥-cyclodextrin is bound differently from maltononaose.At subsite Ϫ3 the cyclodextrin glucose has a position more toward the central residue Tyr-195 (see Fig. 4, A and C), and at subsite ϩ2 the cyclodextrin glucose is bound upside down compared with that in maltononaose (see Fig. 4B).The three remaining glucose moieties of the ␥-cyclodextrin molecule (labeled Ϫ4c, ϩ3c, and ϩ4c) are not bound by any amino acids.The cyclodextrin molecule rests on top of Tyr-195, which does not protrude into the hydrophobic interior of cyclodextrin, in contrast to previous hypotheses (30).An overview of all interactions is presented in Table III and Fig. 5.
Geometry of the CGTase ␥-Cyclodextrin and Maltononaose Ligands-The ␥-cyclodextrin and maltononaose ligands have favorable bond lengths and angles in all sugars (24).In addition, all glucoses assume a favorable 4 C 1 chair conformation, except for those that bind in subsite Ϫ1 (7).However, the internal hydrogen bond network between the glucose OH-2 and OH-3 groups, which is observed in both free cyclodextrins (24,31) and free starch (32), is broken between subsites Ϫ1/ϩ1 and ϩ1/ϩ2 (see Table III and Fig. 5).In the CGTase-maltononaose complex (7), the OH-2/OH-3 network is in addition broken between subsites Ϫ2/Ϫ3, Ϫ4/Ϫ5, and Ϫ5/Ϫ6 but not between ϩ1/ϩ2 (as judged from an O2-O3 distance Ͼ3.3 Å, see Table III and Fig. 5).That these hydrogen bonds are usually present even when malto-oligosaccharides are in aqueous solution (31,33) suggests that CGTase distorts the conformation of maltononaose energetically more than that of ␥-cyclodextrin.
Subsite ϩ2 Binds Two Different Sugar Conformations-At subsite ϩ2, the ␥-cyclodextrin and maltononaose ligands bind in very different conformations, which are both supported by good binding interactions (Table III, Figs.4B and 5).In maltononaose, the glucose at subsite ϩ2 is positioned with its A face (34) toward Phe-183 in an orientation more parallel to Phe-183 than to Phe-259 (gray ring in Fig. 4B).This indicates that the best hydrophobic stacking interactions (34) are made to Phe-183.Furthermore, the glucose O6 atom is pointing toward Tyr-195, while the sugar O3 atom forms a good 2.8-Å hydrogen bond to Lys-232 (Fig. 4B).
In contrast, the ␥-cyclodextrin complex has the subsite ϩ2 glucose residue bound with the other side up.Now the sugar ring A face is oriented parallel and close to Phe-259, indicating good stacking interactions with this residue (Fig. 4B).Interactions with Phe-183 are limited.The cyclodextrin glucose O6 atom is at a 3.3-Å distance from the Lys-232 N atom, but the geometry is not compatible with the presence of a hydrogen bond (Fig. 4B).In summary, at subsite ϩ2, residues Lys-232 and Phe-183 seem to prefer binding to linear substrate, whereas Phe-259 interacts best with cyclodextrin.
Subsite Ϫ3 Binds Two Different Sugar Conformations-Subsite Ϫ3 is also sufficiently promiscuous to provide good contacts with both maltononaose and ␥-cyclodextrin (see Figs. 4A and  5).The binding of maltononaose at subsite Ϫ3 is characterized by a 2.7-Å hydrogen bond between the Asp-371 O␦2 atom and the glucose OH-2 group and by a hydrogen bond between the Asp-196 O␦1 atom and the substrate OH-6 group.The Asp-196 O␦2 atom forms a hydrogen bond with the Tyr-89 O atom (Fig. 4A).This small hydrogen-bonding network is stabilized by the hydrophobic environment provided by Tyr-89, Pro-43, Leu-197, Trp-101, Ile-87, Phe-151 and the glucose at subsite Ϫ4.
In the cyclodextrin complex, the glucose at subsite Ϫ3 is oriented more toward Tyr-195.Residue Asp-196 does not contact the substrate directly but via a water molecule that binds to the glucose atoms O6 (2.7 Å) and O5 (3.1 Å) (Fig. 4A).As in the maltononaose structure, a contact between Asp-371 O␦2 and the glucose OH-2 group is observed, but Asp-371 is displaced by 0.6 Å to accommodate the new binding mode (Fig. 4A).In addition, Arg-47 binds to the O2 and O3 glucose atoms of the cyclodextrin (Fig. 4A).This is the first time that Arg-47 is implicated in direct sugar binding.To make this binding contact, the side chain of Arg-47 has assumed a new conformation compared with unliganded wild type CGTase (15).Thus, at subsite Ϫ3, Arg-47 specifically binds to cyclodextrin, and residue Asp-196 interacts better with a long linear sugar, whereas Asp-371 can support both binding modes.
Ligand-induced Changes in the Main-chain Conformation of CGTase-The main chain of the ␥-cyclodextrin-complexed CG-Tase superimposes very well on that of unliganded CGTase (15) (root mean square deviation, 0.16 Å for 686 C␣ atoms).The only exceptions occur in the loops comprising residues 324 -334 (involved in catalysis around subsite Ϫ1 (7)) and 144 -151 (involved in altered crystal contacts).Thus, to bind a ␥-cyclodextrin, CGTase does not need to adjust its main-chain conformation.
The situation is different when maltononaose-liganded CG-Tase is compared with unliganded CGTase (root mean square deviation, 0.30 Å for 686 C␣ atoms).The relative orientation of the A and E domains has changed slightly, and the sugar binding cleft has narrowed.As in the ␥-CD complex, the loops 144 -151 and 324 -334 have a slightly different conformation.In addition, the loop 190 -199, which contains the important

, where reflection h has intensity I i (h) on occurrence i and mean intensity
where F o and F c are the observed and calculated structure factor amplitudes of reflection h, respectively.
c The free R factor is calculated as the R factor, using F o that were excluded from the refinement (5% of the data).

The Cyclization Mechanism of CGTase
residue Tyr-195, shows a maximal shift of 0.5 Å (see Figs. 3 and  4C).Residues 190 and 199 at both ends of this loop are Ca 2ϩ ion ligands (see Fig. 4C).Another loop, comprising residues 87-93, has been displaced up to ϳ1.3 Å.This loop forms one side of subsite Ϫ3 and shows significantly better electron density in the maltononaose complex than in any other B. circulans strain 251 CGTase complex.Its reorientation might be caused by sugar binding at subsite Ϫ3, since superposition of maltononaose on the unliganded CGTase conformation reveals weak van der Waals repulsions between the glucose C5 and O6 atoms and the Tyr-89 aromatic ring (see Fig. 4A).
A final interesting loop is 175-182, which forms one side of subsites Ϫ5 and Ϫ6 (Fig. 3).It is displaced by ϳ0.7 Å, bringing the main-chain atoms of residues Gly-179 and Gly-180 closer to the bound glucose at subsite Ϫ6 (Fig. 3) (ϳ4 Å).All these loops are intimately connected through various interactions.Thus, in conclusion, maltononaose binding, but not ␥-cyclodextrin binding, causes a clear induced-fit effect in the active site of CGTase.
Ligand-induced Changes in the Conformation of Tyr-195-Together with the main-chain plasticity, the x-ray structures reveal a large conformational flexibility for Tyr-195 (Fig. 4).In the ␥-cyclodextrin-liganded CGTase, the Tyr-195 side chain points toward Phe-183.The Tyr-195 O atom takes part in a three-centered hydrogen bond with the OH-6 and O5 groups of the sugar at subsite ϩ1 (35) (Fig. 4B).However, in the maltononaose complex, Tyr-195 has rotated and shifted 2.6 Å toward subsite Ϫ3 (Fig. 4, B and C).The Tyr-195 O atom now binds to the maltononaose OH-6 group in subsite ϩ1 only.
This reorientation of Tyr-195 and the shift of loop 190 -199 might be caused by a strong steric repulsion at subsite Ϫ6.If maltononaose is superimposed on the unliganded CGTase structure, the backbone carbonyl oxygen of Tyr-195 and the substrate O2 atom at subsite Ϫ6 are at only 2.0 Å distance (Fig. 4C).In addition, the Tyr-195 conformation observed in the maltononaose complex would clash in a ϳ2.0-Å van der Waals contact with a ␥-cyclodextrin ligand at subsite Ϫ3 (Fig. 4C).
This implies that Tyr-195, after being reoriented by maltononaose binding at subsite Ϫ6, is involved in keeping the sugar at subsite Ϫ3 in its observed position.
Ligand-dependent Conformations of Asn-139 and His-140 at Subsite Ϫ1-Although the glucoses of maltononaose and ␥-cyclodextrin in the catalytic site (Ϫ1) occupy similar positions, there is an interesting difference in the conformations of the catalytic site residues His-140 and Asn-139.In cyclodextrinliganded CGTase, the His-140 N␦ atom binds to the carbonyl oxygen of Tyr-100, whereas the His-140 N⑀ atom forms a hydrogen bond to the Asn-139 N␦ atom (purple in Fig. 4C).The Asn-139 O␦ atom acts as a Ca 2ϩ ligand.This His-140 conformation prevents it from binding the Ϫ1 glucose residue of ␥-CD, which is unexpected, because mutagenesis experiments indicate a role for His-140 in sugar binding and transition state stabilization (3,36).Nevertheless, this His-140 -Asn-139 conformation is also observed in unliganded wild type CGTase (15) as well as in inhibitor complexes of CGTase ( 16) and ␣-amylases (28,29).
The situation is different in the maltononaose substrate complex.Here the side chain of Asn-139 has clearly rotated away from Ca 2ϩ , thereby disrupting the hydrogen bond to His-140 (gray in Fig. 4C).In addition, the electron density and hydrogen bond network suggest that the imizadole ring of His-140 is flipped.This enables the 140 N␦ atom to bind the Thr-141 carbonyl oxygen and the 140 N⑀ atom to bind the maltononaose OH-6 group at subsite Ϫ1 (Figs. 4C and 5).This hydrogen bond to the substrate agrees better with the role of His-140 as found from mutagenesis studies (3,36).It suggests that in the maltononaose complex, His-140 is optimally positioned to contribute to catalysis (7).

␥-Cyclodextrin Binds Because Its Degradation Is Sterically
Blocked-To elucidate the atomic basis of the CGTase cyclization reaction, we have determined the first structure of a CG-Tase with a cyclodextrin product bound competently in its active site.This was achieved by using ␥-cyclodextrin (eight glucoses) for soaking instead of ␣or ␤-cyclodextrin (six and seven glucoses, respectively).These latter CDs are degraded by coupling reactions in which the products can bind stably inside the crystal (7,15).However, a coupling reaction with ␥-cyclodextrin would presumably lead to an intermediate that binds at subsite Ϫ8, which is blocked by a crystal contact, thereby forming a kinetic barrier to ␥-cyclodextrin degradation.
The 1.8-Å CGTase-␥-cyclodextrin complex was compared with the 2.1-Å CGTase-maltononaose complex (representing a bound linear substrate ( 7)).Both structures were determined under experimentally identical conditions.These ligands are not stable states of the same cyclization cycle (␥-CD formation requires a linear substrate reaching subsite Ϫ8).Moreover, B. circulans strain 251 CGTase forms ␤-CD in preference to ␥-CD (37).However, cyclization is largely independent of cyclodextrin size, since many amino acids that are typical for CGTase are conserved irrespective of product specificity (see below).Therefore, comparison of both structures can give useful insight into the mechanism of cyclization.
Subsites Ϫ6, Ϫ3, and ϩ2 Contain the Residues Involved in Cyclization-To identify amino acids involved in cyclization, we studied their degree of evolutionary conservation in the active site (Table I).First, some residues are present in all CGTase sequences as well as in Aspergillus oryzae ␣-amylase (which is the ␣-amylase with known structure (38) that is most homolo- gous to CGTase).These residues are predominantly located at subsites Ϫ2, Ϫ1, and ϩ1 and are most likely involved in the catalysis of the bond cleavage reaction (7).Secondly, among the CGTases, some amino acid positions appear to be highly variant, especially at subsite Ϫ7 (Table I).These residues are probably not essential for cyclization but may influence the cyclodextrin product size specificity (13).All other residues, the third class, are those typical for CGTase (bold in Table I) and, therefore, are potentially involved in cyclization.They are mostly located at subsites Ϫ6, Ϫ3, and ϩ2 and also include Tyr-195.Site-directed mutagenesis studies of CGTases, of which an overview is given in Table I, corroborate this view that subsites Ϫ6, Ϫ3, ϩ2, and Tyr-195 are important for cyclization.
Subsite Ϫ6 Selects an Oligosaccharide of Sufficient Length for Cyclization-The first step in cyclization is substrate binding (Fig. 1).At this stage, binding of a sufficiently long oligosaccharide for cyclodextrin formation requires occupation of at least subsites Ϫ1 to Ϫ6 (leading to ␣-cyclodextrin).Thus, the low affinity of subsites Ϫ4 and Ϫ5 could function to prevent binding of too short oligosaccharides.At subsite Ϫ6, the interactions of substrate with Asn-193 and Tyr-167, residues typical for CGTase, and many backbone atoms are strongly conserved (Tables I and III and Fig. 5).Two other CGTase-typical residues at subsite Ϫ6, Gly-179, and Gly-180, have no side chains, since these would sterically block substrate binding.This conservation of subsite Ϫ6 confirms its importance for the selective binding of long oligosaccharide chains.
Subsite Ϫ6 May Activate Catalysis at Subsite Ϫ1-Kinetic studies confirm an increased affinity (K m ) for linear substrates that bind at (and beyond) subsite Ϫ6.However, they also suggest that longer sugar chains have a higher k cat for disproportionation and, thus, are processed more rapidly (39 -41).This could further limit unwanted hydrolysis and disproportionation reactions of small oligosaccharide chains in CGTase.Our x-ray studies suggest that His-140 is involved in such an induced-fit mechanism, since it is specifically activated by binding of maltononaose.
Most likely, the trigger for His-140 activation occurs when substrate binds at subsite Ϫ6 and "pushes" away the carbonyl oxygen of Tyr-195.The subsequent reorientation of Tyr-195 and the loop 190 -199 has two effects.First, it changes the ligation of the nearby Ca 2ϩ ion, which could stimulate reorientation of Asn-139 (Fig. 4C).Second, Tyr-195 forces the glucose at subsite Ϫ3 into its "maltononaose" position, resulting in a rigidification of the amino acids and sugars at subsites Ϫ3 to Ϫ1 (as judged by their low atomic B-factors).Both effects provide a suitable environment for His-140 to assume its optimal orientation for catalysis.
Internal Hydrogen Bonds Promote the Cyclodextrin Binding Mode-After bond cleavage, the next step in cyclization is circularization (Fig. 1).This is intrinsically an energetically unfavorable, "uphill" process, because cyclodextrins have an unfavorable enthalpy of formation (42) and because during circularization the deformed cyclodextrin ring symmetry has to be enforced.An important contribution to circularization could come from the hydrogen bond network between the OH-2 and OH-3 groups of adjacent glucoses, which is formed in the ␥-cyclodextrin complex but not in the maltononaose complex (Table III, Fig. 5B).Completion of this OH-2/OH-3 hydrogen network in the maltononaose structure could be a driving force for the displacement of the nonreducing end toward the acceptor binding sites, as would be necessary in the early stages of circularization.

Tyr-195 Stabilizes an Intermediary Stage in Circularization-Site-directed mutagenesis experiments indicate that
Tyr-195 is very important for cyclization, especially its aromatic moiety (Table I) (37).However, the x-ray structures show only hydrogen bond interactions between Tyr-195 and maltononaose or ␥-cyclodextrin (Table III and Fig. 5).This suggests that the aromatic ring of Tyr-195 has a role during circularization.From the x-ray structures it appears that in the maltononaose complex Tyr-195 exposes, together with Phe-183, exposes a hydrophobic surface (Fig. 4B).This could function as "greasy slide" (14,43) to facilitate movement of the sugar chain end during circularization.Interestingly, when the glucose at subsite Ϫ3 assumes a ␥-cyclodextrin-like conformation, it sterically clashes with this conformation of Tyr-195, causing the dismantling of the greasy slide in the final stages of circularization.
Subsite Ϫ3 Catalyzes the Transition from Linear to Circular Sugar Chains-During circularization, the sugar at subsite Ϫ3 has to change from a maltononaose binding mode, stabilized by Asp-196 and Asp-371, into a ␥-cyclodextrin binding mode, stabilized by Arg-47 and Asp-371 (Fig. 4A).All these residues are conserved in CGTases, except for Arg-47, which is sometimes replaced by Lys or His.This conserved basicity might preserve a charge-charge interaction with Asp-371 (at ϳ5 Å), which stabilizes the conformation of subsite Ϫ3.In addition, the nonconserved residues Tyr-89 and Asn-94 (at ϳ5 Å from the sugar) are part of subsite Ϫ3, as shown by mutagenesis studies (Table I).
When the interactions of Arg-47, Tyr-89, Asp-196, or Asp-371 with carbohydrates are removed by site-directed mutagenesis, not only do the binding affinities (K m ) change for linear substrates or cyclodextrins, but a 4 -10-fold decrease of the k cat for cyclization and the reverse cyclodextrin degradation, or coupling, reaction results. 2,4This indicates that, in addition to their role in substrate and product binding, residues at subsite Ϫ3 are actively involved in circularization.This important function of subsite Ϫ3 partly derives from its strategic position between the conserved subsites Ϫ1 and Ϫ2 (Table I) and the weakly binding subsites Ϫ4 and Ϫ5 (Table III).If the sugar orientation is changed at subsite Ϫ3, a large influence on the positions of sugars at more distant subsites is seen.
At Subsite ϩ2 Phe-259 Stabilizes the Cyclodextrin Binding Mode-In the later stages of circularization, the circular sugar chain reaches subsite ϩ2, where it competes with free sugar acceptors (e.g.maltose) for binding.The x-ray structures indicate that Phe-183 and Lys-232 at subsite ϩ2 preferably interact with such linear acceptors, which is consistent with the presence of homologous residues in ␣-amylases (Table I).Nevertheless, mutagenesis of Phe-183 indicates a critical role in cyclization (30).Possibly, Phe-183 is correctly positioned to guide the circularizing movement of the linear chain toward the acceptor sites.The x-ray structures further show that Phe-259 at subsite ϩ2, a typical residue for CGTases, selectively stabilizes cyclodextrins.This is supported by mutants in Phe-259, which show a greater loss in affinity (as judged by the K m value) for cyclodextrins than for maltose (30).Thus Phe-259 stimulates the use of a circularized sugar chain as the acceptor instead of a free sugar.Conclusions-The active site of CGTase is an interesting example of an architecture in which a particular specificity is added to a conserved ␣-amylase bond cleavage machinery.Sequence comparisons (Table I) indicate that this bond cleavage machinery is located at subsites Ϫ2, Ϫ1, and ϩ1, whereas the cyclization specificity is confined to subsites ϩ2, Ϫ3, Ϫ6, and the centrally located residue Tyr-195.Our analysis of the structures of B. circulans strain 251 E257Q/D229N CGTase, in The Cyclization Mechanism of CGTase complex with maltononaose and ␥-cyclodextrin, confirms this picture.Maltononaose and ␥-cyclodextrin are bound in similar ways at subsites Ϫ2, Ϫ1, and ϩ1 but show conformational differences at subsites ϩ2 and Ϫ3.At those latter subsites, specific "cyclodextrin binding" residues exist (Phe-259 and Arg-47) as well as "linear substrate binding" residues (Phe-183, Lys-232, and Asp-196).These separate functions can be exploited to engineer enzymes with specific properties.At subsite Ϫ6, the role of Asn-193 and Tyr-167 seems to be selecting a substrate of sufficient length for cyclodextrin formation (minimally 6 glucoses).Finally, the aromatic ring of Tyr-195 could bind an intermediary stage in circularization, since it is important for cyclization activity, but does not interact strongly with either linear substrate or cyclodextrin.

FIG. 2 .
FIG. 2. Stereo picture of the final A -weighted 2 F o ؊ F c electron density that defines the ␥-cyclodextrin bound in the CGTase active site.The density was contoured at 1 unit S.D. (1).The labels indicate subsite nomenclature.

FIG. 4 .
FIG. 4. Close-ups of the ␥-cyclodextrin structure (yellow and red, with corresponding amino acids in purple) superimposed on the maltononaose structure (7) (gray and white).A, detail of subsite Ϫ3.The conformation of Arg-47 in both structures differs from wild type, unliganded CGTase (not shown).B, detail of subsite ϩ2; for explanation see text.C, rearrangements around His-140 and the loop 190 -199.For clarity the cyclodextrin is only drawn at subsites Ϫ1 to Ϫ3.

FIG. 5 .
FIG. 5. Overview of the interactions between CGTase and maltononaose (7) (A) or ␥-cyclodextrin (B).The distances associated with the interactions are in TableIII.For clarity, not all interactions at subsites Ϫ2, Ϫ1, and ϩ1 are shown.Symm rel.contacts, contacts made to a symmetry-related CGTase molecule in the crystal.

TABLE I
The function of residues in CGTase sugar binding subsites

TABLE III
Interactions between substrate, product, and CGTaseFor an overview of the subsites see Figs.3 and 5. WM is shorthand for water-mediated.NA indicates a nonappropriate subsite label.-indicates the absence of an interaction.