Keiko Haga

The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping-Pong mechanism

A new photometric assay of the disproportionation activity of cyclodextrin glucanotransferase (CGTase) using 3‐ketobutylidene‐β‐2‐chloro‐4‐nitrophenyl ‐maltopentaoside as the donor, proved that the transglycosylation reaction of CGTase was operated by a Ping‐Pong Bi Bi mechanism. The values of the k cat/K m acceptor proved that the same configurations of free hydroxyl groups with those of d‐glucopyranose at C2, C3 and C4 positions were required for the acceptors used by CGTase. The structure around C6 on acceptors was not essential for acceptor function, but it was recognized by CGTase, since the values of k cat/K m for d‐xylose were smaller than that for d‐glucose. The value of k cat/K mfor maltose was about 20‐times larger than that for d‐glucose, indicating that at least two glucopyranosyl rings are recognized by the acceptor binding sites.

Functional relationships between cyclodextrin glucanotransferase from an alkalophilic Bacillus and α-amylases : site-directed mutagenesis of the conserved two Asp and one Glu residues

Comparison of the amino acid sequences of cyclodextrin glucanotransferases (CGTases) with those of α‐amylases revealed that two Asp and one Glu residues, which are considered to be the catalytic residues in α‐amylases, were also conserved in CGTases. To analyze the function of the three conserved amino acid residues in CGTases, site‐directed mutagenesis was carried out. The three mutant CGTases, in which Asp229, Glu257 and Asp328 were individually replaced by Asn or Gln, completely lost both their starch‐degrading and β‐cyclodextrin‐forming activities, whereas another mutant CGTase, in which Glu264 replaced by Gln, retained these activities. The three inactive enzymes retained the ability to be bound to starch. These results suggest that Asp229, Glu257 and Asp328 play an important role in the enzymatic reaction catalyzed by CGTase and that a similar catalytic mechanism is present in both CGTases and α‐amylases.

X-ray structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011. Comparison of two independent molecules at 1.8 A resolution.

Cyclodextrin glucanotransferase (CGTase) is an enzyme which produces cyclodextrins by the degradation of starch. The enzyme from alkalophilic Bacillus sp. 1011, consisting of 686 amino acid residues, was crystallized from the solution containing 20% PEG 3000 and 20% 2-propanol at pH 5.6 adjusted with citrate buffer. The space group was P1 and the unit cell contained two molecules (V(m) = 2.41 A(3) Da(-1)). The structure was solved by the molecular replacement method and refined to a conventional R value of 0.161 (R(free) = 0.211) for the reflections in the resolution range 1.8-10 A by energy minimization combined with simulated annealing. The molecule consists of five domains, designated A-E, and its backbone structure is similar to the structure of other bacterial CGTases. The molecule has two calcium binding sites where calcium ions are coordinated by seven ligands, forming a distorted pentagonal bipyramid. The two independent molecules are related by a pseudotwofold symmetry and are superimposed with an r.m.s. deviation value of 0.32 A for equivalent C(alpha) atoms. Comparison of these molecules indicated the relatively large mobility of domains C and E with respect to domain A. The active site is filled with water molecules forming a hydrogen-bond network with polar side-chain groups. Two water molecules commonly found in the active center of both molecules link to several catalytically important residues by hydrogen bonds and participate in maintaining a similar orientation of side chains in the two independent molecules.

Crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 determined at 1.9 A resolution.

The crystal structure of asparagine 233‐replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 was determined at 1.9 Å resolution. While the wild‐type CGTase from the same bacterium produces a mixture of mainly α‐, β‐ and γ‐cyclodextrins, catalyzing the conversion of starch into cyclic or linear α‐1,4‐linked glucopyranosyl chains, site‐directed mutation of histidine‐233 to asparagine changed the nature of the enzyme such that it no longer produced α‐cyclodextrin. This is a promising step towards an industrial requirement, i.e. unification of the products from the enzyme. Two independent molecules were found in an asymmetric unit, related by pseudo two‐fold symmetry. The backbone structure of the mutant enzyme was very similar to that of the wild‐type CGTase except that the position of the side chain of residue 233 was such that it is not likely to participate in the catalytic function. The active site cleft was filled with several water molecules, forming a hydrogen bond network with various polar side chains of the enzyme, but not with asparagine‐233. The differences in hydrogen bonds in the neighborhood of asparagine‐233, maintaining the architecture of the active site cleft, seem to be responsible for the change in molecular recognition of both substrate and product of the mutant CGTase. Copyright

Crystal Structure of Cyclodextrin Glucanotransferase from Alkalophilic Bacillus sp. 1011 Complexed with 1-Deoxynojirimycin at 2.0 Å Resolution

1-Deoxynojirimycin, a pseudo-monosaccharide, is a strong inhibitor of glucoamylase but a relatively weak inhibitor of cyclodextrin glucanotransferase (CGTase). To elucidate this difference, the crystal structure of the CGTase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin was determined at 2.0 A resolution with the crystallographic R value of 0.154 (R(free) = 0.214). The asymmetric unit of the crystal contains two CGTase molecules and each molecule binds two 1-deoxynojirimycins. One 1-deoxynojirimycin molecule is bound to the active center by hydrogen bonds with catalytic residues and water molecules, but its binding mode differs from that expected in the substrate binding. Another 1-deoxynojirimycin found at the maltose-binding site 1 is bound to Asn-667 with a hydrogen bond and by stacking interaction with the indole moiety of Trp-662 of molecule 1 or Trp-616 of molecule 2. Comparison of this structure with that of the acarbose-CGTase complex suggested that the lack of stacking interaction with the aromatic side chain of Tyr-100 is responsible for the weak inhibition by 1-deoxynojirimycin of the enzymatic action of CGTase.

Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707

Maltohexaose‐producing amylase (G6‐amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short‐chain amylose (DP = 17). Our previous crystallographic study showed that G6‐amylase has nine subsites, from −6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6‐amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6‐amylase complexes with G6 and maltopentaose (G5). In the active site of the G6‐amylase/G5 complex, G5 is bound to subsites −6 to −2, while G1 and G6 are found at subsites + 2 and −7 to −2, respectively, in the G6‐amylase/G6 complex. In both structures, the glucosyl residue located at subsite −6 is stacked to the indole moiety of Trp140 within a distance of 4Å. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short‐chain amylose by W140L is lower than that by W140Y or wild‐type enzyme. The face‐to‐face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite −6 and to govern product specificity for G6 production.

Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site.

Cyclodextrin glycosyltransferase (CGTase) belonging to the α‐amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related α‐1,4‐glucans. The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in α‐amylase. To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose‐complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y). The temperature factors of the region 259–269 in native F283L increased >10 Å2 compared with the wild type. The complex formation with acarbose not only increased the temperature factors (>10 Å2) but also changed the structure of the region 257–267. This region is stabilized by interactions of Phe283 with Phe259 and Leu260 and plays an important role in the cyclodextrin binding. The conformation of the side‐chains of Glu257, Phe259, His327, and Asp328 in the catalytic site was altered by the mutation of Phe283 with leucine, and this indicates that Phe283 partly arranges the structure of the catalytic site through contacts with Glu257 and Phe259. The replacement of Phe283 with tyrosine decreased the enzymatic activity in the basic pH range. The hydroxyl group of Tyr283 forms hydrogen bonds with the carboxyl group of Glu257, and the pKa of Glu257 in F283Y may be lower than that in the wild type.

Crystal Structure of Alkalophilic Asparagine 233-Replaced Cyclodextrin Glucanotransferase Complexed with an Inhibitor, Acarbose, at 2.0 å Resolution

The product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces alpha-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly alpha-, beta-, and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 A resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like a-cyclodextrin.