THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 37, Issue of September 10, pp. 26279 –26286, 1999 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A. Crystal Structure of a Maltogenic Amylase Provides Insights into a
Catalytic Versatility*

(Received for publication, April 13, 1999, and in revised form May 25, 1999) Jeong-Sun Kim‡, Sun-Shin Cha‡, Hyun-Ju Kim‡, Tae-Jip Kim§, Nam-Chul Ha‡, Sang-Taek Oh‡,
Hyun-Soo Cho‡, Moon-Ju Cho‡, Myo-Jeong Kim§, Hee-Seob Lee§, Jung-Wan Kim¶,
Kwan Yong Choi‡, Kwan-Hwa Park§, and Byung-Ha Oh‡
From the Department of Life Science, and School of Environmental Engineering, Pohang University of Science andTechnology, Pohang, Kyungbuk, 790-784, South Korea, the §Department of Food Science and Technology & ResearchCenter for New Bio-Materials in Agriculture, Seoul National University, Suwon, 441-744, South Korea, the Departmentof Biology, University of Inchon, Inchon, 402-749, South Korea, and the iCenter for TARA, University of Tsukuba,305 Tsukuba, Japan Amylases catalyze the hydrolysis of starch material
Starch is the main source of energy for a wide variety of and play central roles in carbohydrate metabolism.
living organisms. It is the polymer of glucose, linked by either Compared with many different amylases that are able to
D-(1,4)- or a-D-(1,6)-glycosidic bonds. Starch is hydrolyzed by hydrolyze only a-D-(1,4)-glycosidic bonds, maltogenic
several different types of hydrolytic enzymes widespread in amylases exhibit catalytic versatility: hydrolysis of a-D-
nature, most of which can be grouped in the a-amylase family (1,4)- and a-D-(1,6)-glycosidic bonds and transglycosyla-
(1). Amylases are classified according to their enzymatic action tion of oligosaccharides to C3-, C4-, or C6-hydroxyl
pattern. Glucoamylases (EC and b-amylases (EC groups of various acceptor mono- or disaccharides. It are exo-type enzymes cleaving glucose and maltose has been speculated that the catalytic property of the
units, respectively, from the nonreducing end of starch mate- enzymes is linked to the additional ;130 residues at the
rials by hydrolyzing a-D-(1,4)-glycosidic bonds. a-Amylases (EC N terminus that are absent in other typical a-amylases. are endo-type enzymes catalyzing the cleavage of the The crystal structure of a maltogenic amylase from a
internal a-D-(1,4)-glycosidic bonds of starch, glycogen, and var- Thermus strain was determined at 2.8 Å. The structure,
ious oligosaccharides. Pullulanases (EC cleave the an analytical centrifugation, and a size exclusion col-
umn chromatography proved that the enzyme is a dimer
D-(1,6)-glycosidic bonds of the substrate pullulan, in solution. The N-terminal segment of the enzyme folds
which is the polymer of maltotriose linked by a-D-(1,6)-glyco- into a distinct domain and comprises the enzyme active
site together with the central (a/b) barrel of the adja-
Several groups of starch-hydrolyzing enzymes are known to cent subunit. The active site is a narrow and deep cleft
harbor more than single enzyme activity. One group of these, suitable for binding cyclodextrins, which are the pre-
maltogenic amylases (MAases;1 EC exhibit unique ferred substrates to other starch materials. At the bot-
characteristics that are different from other a-amylases (2–5) tom of the active site cleft, an extra space, absent in the
in that they exhibit (i) a dual activity of a-D-(1,4)- and a-D-(1,6)- other typical a-amylases, is present whose size is com-
glycosidic bond cleavages; (ii) an activity of a-D-(1,4)- to a-D- parable with that of a disaccharide. The space is most
(1,3)-, a-D-(1,4)-, or a-D-(1,6)-transglycosylation; and (iii) an likely to host an acceptor molecule for the transglycosy-
activity of cleaving acarbose, a pseudo-tetrasaccharide compet- lation and to allow binding of a branched oligosaccha-
itive inhibitor of a-amylases. The enzymes prefer cyclodextrins ride for hydrolysis of a-D-(1,4)-glycosidic or a-D-(1,6)-gly-
(CDs) to starch or pullulan as substrates in that the hydrolysis cosidic bond. The (a/b) barrel of the enzyme is the
of b-CDs (seven glucose units) is ;100 times faster than that of preserved scaffold in all the known amylases. The struc-
starch or pullulan (5). In contrast, the other a-amylases with ture represents a novel example of how an enzyme ac-
the single hydrolysis activity cannot hydrolyze CDs or pullulan.
quires a different substrate profile and a catalytic ver-
MAases cleave a-D-(1,4)-glycosidic bond much more efficiently satility from a common active site and represents a
than a-D-(1,6)-glycosidic bond. The enzymes hydrolyze CDs and framework for explaining the catalytic activities of
starch mainly to maltose at low concentration of the substrates, transglycosylation and hydrolysis of a-D-(1,6)-glycosidic
thereby they are named "maltogenic amylase". Different sugar molecules, including glucose, fructose, maltose, and cellobiose,can serve as acceptors for the transglycosylation. Comparedwith cyclodextrin glycosyltransferases (CGTases; EC, * This study made use of the x-ray Facility at Pohang Light Source which catalyze the formation of CDs from starch by transgly- and of BL6B and BL18B beamlines at PF in Japan and was supported cosylation, the transglycosylation activity of MAases is much by the Research Center for New Bio-Materials in Agriculture, Seoul more inefficient because they convert the substrates mainly to National University, and in part by Grant HMP-98-F-5-0017 of GoodHealth 21 Program, MHW, and by the Sakabe Project of TARA. The hydrolysis products. The property, if not all, is shared by other costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked "adver-tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate 1 The abbreviations used are: MAase, maltogenic amylase; ThMA, Thermus maltogenic amylase; BSMA, B. stearothermophilus malto- The atomic coordinates and structure factors (code 1SMA) have been genic amylase; CGTase, cyclodextrin glycosyltransferase; NPase, deposited in the Protein Data Bank, Research Collaboratory for Struc- neopullulanase; CDase, cyclomaltodextrinase; CD, cyclodextrin; PTS, tural Bioinformatics, Rutgers University, New Brunswick, NJ (http:// pseudo-trisaccharide; MIR, multiple isomorphous replacement; MR, molecular replacement; BES, N,N-[bis(2-hydroxyethyl)amino]ethane- ** To whom correspondence should be addressed. Tel.: 82-562-279- sulfonic acid; NCS, noncrystallographic symmetry; TVAII, T. vulgaris 2289; Fax: 82-562-279-2199; E-mail: [email protected].
R-47-amylase II.
This paper is available on line at http://www.jbc.org
Crystal Structure of Maltogenic Amylase FIG. 1. Sequence alignment of ThMA (GenBank™ accession number O069007), BSMA (Q45490), B. stearothermophilus NPase
(P38940), Bacillus sp. CDase (O82982), and A. oryzae TAKA-amylase (P10529). The secondary structure assignment and numbering at the
top of the alignment correspond to ThMA. In the secondary structure notations, Nb1, Bb1, and Cb1 represent the first b-strand of the N-, (a/b)8
barrel-, and C-domain, respectively, and so on. Amino acids that are not conserved, compared with the ThMA sequence, are lightly shaded. The
black boxes represent the catalytic residues. Glu-332 is indicated by an asterisk whose substitution with histidine severely affects the transgly-
cosylation activity of ThMA.
amylolytic enzymes with different names, including neopullu- exhibits some extent of sequence homology with the smaller lanases (NPases; EC (6, 7) and cyclomaltodextri- a-amylases and contains invariant catalytic residues (1). Un- nases (CDases; EC (8, 9), both of which are homolo- like other starch-hydrolyzing enzymes, the three groups of the gous to maltogenic amylases with sequence identity of 40 – enzymes are intracellular enzymes. In Klebsiella oxytoca, the 86%. The three groups of amylases are high molecular weight open reading frames for CDase and an extracellular enzyme amylases because of a unique addition of about 130 residues at CGTase are clustered on the chromosomal DNA together with the N terminus compared with the other a-amylases containing genes coding for products homologous to the maltose and linear the single activity of hydrolyzing a-D-(1,4)-glycosidic bonds.
maltodextrin uptake system (10). The finding suggested a The C-terminal region of the enzymes (about 490 amino acids) starch degradation pathway where CDase is involved in the Crystal Structure of Maltogenic Amylase Structure determination and crystallographic statistics (outer shell) (%)a Phasing power (acen/cen) Figure of merit (20–3.1 Å)b (acen/cent/all) Number of refined atoms, Protein/water/erythritol r.m.s.deviation bond lengths/angles 2 I u/SI b Figure of merit 5 ,uSP(a)eia/SP(a)u., where a is the phase and P(a) is the phase probability distribution.
c R-factor 5 SiF u 2 uF i/SuF u, where uF u and uF u are the observed and calculated structure factor amplitudes respectively.
was calculated with 5% of the data.
intracellular degradation of CDs that are generated and trans-ported into the cell by CGTase and by a specific uptake system,respectively.
The crystal structures of a-amylases from many different sources (Kadziola et al. (11) and Fujimoto et al. (12), and ref-erences therein) revealed that this family of proteins has a common folding scaffold, the (a/b) barrel, and that the active site is located on top of the barrel. However, structural infor-mation of MAases has been lacking. We have determined thestructure of maltogenic amylase from a Thermus strain (ThMA). ThMA shows optimum temperature of 60 °C, which ishigher than that of any other maltogenic or similar enzymereported so far. The structure reveals how the unique ;130extra residues at the N terminus modify the common active sitepreserved throughout the a-amylase family to achieve the dis-tinct property of the enzyme.
MATERIALS AND METHODS Crystallization—Initial effort was put into the structure determina- tion of Bacillus stearothermophilus maltogenic amylase (BSMA) whosecrystallization condition we have reported (13). We suffered because offragility of the crystals, especially in the presence of a heavy metalcompound, and subsequently switched to the crystallization of ThMAwhich led to the structure determination of the both enzymes. Genecloning and overproduction of ThMA was described recently (5). TheThMA crystals were obtained by vapor diffusion from droplets contain-ing 2 ml of protein solution (10 mg/ml in 20 mM maleate, pH 6.8) plus 0.5 ml of precipitant solution containing 0.5 M lithium sulfate, 0.3 M ammo-nium sulfate, 0.1 M sodium citrate (pH 5.6), and 4% ethanol (v/v), whichwere equilibrated against 1 ml of the same precipitant solution at 22 °C.
The crystals belong to the space group P6 , with cell dimensions of a 5 b 5 118.04 Å, c 5 266.88 Å, and contain two molecules in the asym-metric unit.
X-ray Data Collection, Structure Determination, and Refinement— Because of the large unit cell dimensions of ThMA crystals, all x-raydiffraction data were collected from flash-cooled crystals by using syn-chrotron radiation from TARA and BL18B beamlines at Photon Fac-tory, Japan. The cryoprotectant solution contained 0.5 M lithium sul-fate, 0.3 M ammonium sulfate, 0.1 M BES (pH 6.5), 4% ethanol (v/v), andsaturated erythritol. Data reduction, merging, and scaling were accom-plished with the programs DENZO and SCALEPACK (14). Whilesearching for heavy atom derivative crystals, the (a/b) structures of FIG. 2. a, size exclusion column chromatography of ThMA and size various amylases were used as search models in the MR (molecular marker proteins. Superdex 200 HR10/30 column (Amersham Pharma- replacement) method. Low sequence homologies of ThMA to other typ- cia Biotech) was used. The proteins were eluted with 50 mM phosphatebuffer (pH 7.0) containing 150 mM NaCl. The flow rate was 0.4 ml/min.
The inset shows the line fitting of the elution time versus logarithm ofthe molecular weight of the size markers. —, ThMA; z z z z , standard 1 concentration distribution of the protein as a function of the square of (b-amylase:200,000 1 carbonic anhydrase:29,000); 2 2 2, standard 2 the radial position is shown. The partial specific volume of ThMA was (alcohol dehydrogenase:150,000 1 cytochrome c:12,400); 2 z z 2, stand- calculated as 0.7354 cm3/g from the amino acid sequence of the protein.
ard 3 (bovine serum albumin:66,000). b, equilibrium centrifugation of The solid line indicates the calculated curve for dimeric species. R2, the ThMA. The concentration of ThMA was 0.087 mg/ml in 50 mM sodium coefficient of determination, indicates an excellent fit to an ideal di- acetate buffer (pH 6.0). The equilibrium was attained in 40 h. The meric species model.
Crystal Structure of Maltogenic Amylase FIG. 3. a, stereoview of Ca tracings of the ThMA monomer. Every other 20th residue positions are numbered. The drawing does not contain the Ca positions for residues 160 –163, 259 –264, and 277–280. b, the ribbon diagram of the ThMA monomer. The N-, (a/b) barrel, and C-domain are in green, cyan, and yellow, respectively. c, the ribbon diagram of the ThMA dimer in complex with a computationally docked b-CD, which is themost preferred substrate for the maltogenic and related enzymes. The two subunits, related by the molecular 2-fold axis lying on the figure, arelabeled with different colors. The docking of the substrate molecule did not require reorientation of the protein amino acid side chains and changein torsion angles of substrate. The model of b-CD bound to soybean b-amylase (27) was used without modification.
ical a-amylases prevented us from obtaining a correct solution. To our try-related molecules. Furthermore, the phase derived from MR solu- surprise, the (a/b) structure of b-amylase (Protein Data Bank code tions identified a holmium position in a Fourier difference map which 1BYA) as a search model, which bears no meaningful sequence homol- coincides with the position located from 3.0 Å resolution isomorphous ogy to ThMA, yielded correct positions of the two molecules in the Patterson difference map. The successful MR demonstrates that initial asymmetric unit with CCP4 version of AMoRe (15) (for the first solu- phase information can be derived using available structures coupled tion) and with X-PLOR (16) (for the second solution). The translation with a correct prediction of the tertiary folding pattern of an interested functions were calculated with the highest peak with a correlation of protein. However, we do not understand why the (a/b) barrel of TAKA- 7.3% in a rotation search using AMoRe. The search found the highest amylase A, which should be more homologous to ThMA than the b-am- peak with a correlation of 10.8% and an R-factor of 56.5%. The increase ylase (see below), failed to give a correct solution.
in the correlation coefficient suggested that the peak was a correct It was not at all straightforward to solve the structure by using the solution. However, efforts to find the position of the second molecule phase information derived from MR, and additional phase information after fixing the position of the first solution did not yield a promising was obtained from three heavy atom derivative crystals (Table I) by solution. A self-rotation search with X-PLOR yielded a very strong peak MIR (multiple isomorphous replacement) method. Heavy atom binding for f 5 7.5, c 5 0, k 5 180. The first solution was rotated according to sites were identified by Fourier difference analysis using the phases the noncrystallographic symmetry (NCS), and a translation search derived from MR. The heavy atom positions were used to calculate MIR along the xy plane was performed, which was followed by a translation phases. The MIR phases with the three derivative data had a mean search along the z-axis to correlate the relative z-positions of the two figure of merit of 0.47 at 3.1 Å resolution and were improved by solvent solutions. The calculations generated the final solutions with an R- flattening and by 2-fold NCS density averaging. Some parts of the factor of 49.9% after a rigid body refinement. When examined on a partial model inconsistent with the MIR map were further truncated. A graphics computer, the two molecules showed no overlap with symme- combination of the partial model and the MIR phases and a 2-fold NCS Crystal Structure of Maltogenic Amylase rithm (19). The N-terminal 129 amino acids and the C-terminal;30 amino acids in the former three enzymes are completelyabsent in TAKA-amylase A. The central region exhibits a lowdegree of sequence homology, but the three catalytic residues,Asp-328, Glu-357, and Asp-424 (ThMA numbering) are invari-ant. The structure of ThMA can be divided into three distinctdomains, the N-domain (residues 1–124) composed of b-strandsexclusively, the central (a/b) barrel domain, and the C-domain composed of eight b-strands (residues 505–588) (Fig. 3). Super-position of the structures of ThMA and TAKA-amylase A re-veals that the C-domain as well as the (a/b) barrel of the two enzymes are structurally conserved in contrary to the sequencealignment. However, a structural domain corresponding to theN-domain of ThMA is totally absent in the structure of TAKA-amylase A. It is a b-sandwich-like structure composed of 12anti-parallel b-strands (Fig. 3). The domain is distinctivelyseparated from the central body of the ThMA structure, but itis involved in extensive interactions with the (a/b) domain of the adjacent molecule in the asymmetric unit. A total of 1390 Å2 in solvent accessible surface of one molecule is excluded bythe interactions, which is an indication of a dimer formation ofthe enzyme in solution. More than two-thirds of the excludedsurface is hydrophobic surface. The observation led us to runThMA on a size exclusion column to find that the enzyme waseluted as if the apparent molecular weight of the enzyme is; 130,000, close to the calculated dimeric molecular weight (136, 414) (Fig. 2a). An ultracentrifugation analysis furtherevidenced the dimerization of ThMA in solution as shown inFig. 2b. BSMA exhibits the same chromatographic profile andis also dimeric with tertiary and quaternary structures nearly identical to those of ThMA. Therefore, it is firmly established FIG. 4. Schematic drawings of products from reaction of acar-
that MAases are dimeric in solution. Very recently, the crystal bose with ThMA. Acarbose is hydrolyzed to PTS and glucose. Three
structure of a related enzyme, Thermoactinomyces vulgaris different products can be generated by transglycosylation of PTS toglucose. One of those generated by a-D-(1,4)-transglycosylation is acar- R-47-amylase II (TVAII) hydrolyzing cyclodextrins and pullu- bose. The activity of a-D-(1,4)-transglycosylation can be detected by lan, was reported (20) which shows 44% sequence identity to using an acceptor molecule such as a-methylglucoside.
ThMA. The overall structure of TVAII is very similar to that ofThMA. Two molecules of TVAII were also contained in the averaging at 8 –3.1 Å resulted in a significantly improved electron asymmetric unit of the crystals of the enzyme (space group density map in which many regions could be traced. The MIR phasingand density modifications were carried out using the CCP4 suite (17).
P2 2 2 ). The two molecules exhibit the same molecular con- Crystallographic refinements, iterative map calculations, and model tacts as the ThMA dimer does. Although the oligomerization buildings were done using X-PLOR, CCP4 suite, respectively. The 2-fold state of TVAII was not investigated, the cumulative evidence NCS restraints were maintained until the last refinement. From the strongly suggest that TVAII and other homologous enzymes beginning of the refinement, 5% of total reflections from the native data classified as CDases and NPases should be dimeric in solution.
set were set aside for monitoring R value. The final model at 2.8 Å resolution consists of 574 amino acids with an R value of 20.9% and an Therefore, the substrate profile and catalytic property of these of 27.0%. The model does not contain highly disordered residues enzymes would be best explained by the active site configura- 160 –163, 259 –264, and 277–280 for which electron densities are tion in the dimeric state.
The N-domain Modifying the Common Active Site Struc- The refined dimeric structure of ThMA was used to determine the ture—The consequence of the dimer formation of ThMA is structure of BSMA by MR, which shares 69% sequence identity toThMA. Contrary to our earlier prediction that the asymmetric unit of striking. The N-terminal domain of one subunit covers a part of BSMA crystals would contain three to four molecules (13), only one the top of the (a/b) barrel of the other subunit, which corre- dimer is present with a high solvent content of 70.1%. The BSMA sponds to the active site cleft of all the known a- or b-amylases.
structure is very similar to that of ThMA and is not discussed in detail As a result, the dimer interface at the top of the barrel forms a in this report.
narrow and deep groove that is ;17 Å in length, ;8 Å in width, Analytical Ultracentrifugation—Sedimentation equilibrium meas- and ;18 Å in depth, distinctively different from the wide and urements were performed at 20 °C on a Beckman Optima XL-A analyt-ical ultracentrifuge, using a four-hole rotor with standard double sector shallow active site clefts of the smaller a-amlyases. The groove cell at a rotor speed of 10,000 rpm. The values of the two variables, must be the active site cleft because the three invariant cata- absorbances at 280 nm versus radial positions were obtained. The lytic residues are located at the bottom of the groove. They are apparent molecular weight of ThMA was calculated by fitting data sets found in the same relative position in space as the correspond- to a single species model using the software BMPD-Nonlinear regres- ing residues in TAKA-amylase A. The shape of the active site sion. The partial specific volume of ThMA and solvent density werecalculated as described by Zamyatnin (18).
cleft of ThMA explains much slower hydrolysis of starch thanCDs by the enzyme. The ring structures of CDs should be narrower in width than that of starch segment (amylose), ThMA Is a Homodimer—Fig. 1 shows sequence alignments which assumes a coiled helical structure composed of six, of MAases, NPase, CDase, and Aspergillus oryzae TAKA-amy- seven, or eight glucose residues per turn of the helix in aqueous lase A, which is listed as the most homologous smaller a-amy- solution (21). A CD fits into the groove snugly as it can be lase in a sequence alignment search using the BLAST algo- represented by a hypothetical binding mode of b-CD, docked Crystal Structure of Maltogenic Amylase FIG. 5. Extra sugar-binding space at the active site of ThMA. a, molecular surfaces of ThMA and a model-built maltose at the extra
sugar-binding space. In this model, the protein and docked b-CD atom positions were held fixed, and the torsion angle of the maltose glycosidicbond was rotated until the "solid docking" in program QUANTA allowed the favorable docking. The docking did not require reorientation of theprotein side chains. b, stereo view of the active site of ThMA with hypothetical binding modes of the substrate b-CD (in coral) and maltose (in lightblue). The position of b-CD and maltose is the same as in Fig. 3c and in Fig. 5a, respectively. The active site residues (Asp-328, Glu-357, Asp-424)plus Glu-332, identified as important for transglycosylation, are labeled. All oxygen atoms are in red. Several other residues (His-205, Tyr-207,Phe-289, Trp-359, His-423, Asp-468, Pro-469, and Trp-47 from the adjacent subunit) in close contact with the modeled b-CD are shown withoutlabels. The segments of the (a/b) barrel (from one subunit) and the N-domain (from the other subunit) comprising the active site cleft are shown in green and magenta. Fig. 3 and Fig. 5, a and b, were prepared using the programs MOLSCRIPT (28), GRASP (29), and QUANTA, respectively.
computationally into the groove (Fig. 3c) using the "solid dock- branched molecule by the presence of a-D-(1,6)-glycosidic bond, ing" module in QUANTA (Molecular Simulation, Inc.). The would not reach the catalytic residues as easily as CDs either.
program allows a successful docking only when an electrostatic An Extra Sugar-binding Space at the Bottom of the Catalytic and geometric complementarity is accomplished between a Site—Unlike CGTase, ThMA mainly cleaves glycosidic bonds host and a guest molecule. The deep groove is well occupied by at the beginning of the enzyme reaction, and transglycosylation the ring structure of the b-CD and many residues from both the products are detected after a time lag when cleavage products (a/b) barrel and N-terminal domains are involved in favorable are accumulated. The observation raises a possibility that the interactions with the modeled b-CD as shown in a close-up transglycosylation by ThMA is the reverse reaction of the hy- view of the substrate-binding model (Fig. 5). At the bottom of drolysis driven by a high concentration of small oligosaccharide the cleft, one of the glycosidic bonds of the b-CD is located close products. We established that it is not the case for ThMA.
to the catalytic residues. The catalytic residues of ThMA would Acarbose, a pseudo-tetrasaccharide that acts like a transition- be reached only by the disordered part of starch. Pullulan, a state analogue with a-amylases, is cleaved by ThMA to produce Crystal Structure of Maltogenic Amylase FIG. 6. Proposed mechanism for competition of transglycosylation and hydrolysis reaction at the active site of ThMA. A proposal
for a double-displacement reaction is followed. The third conserved residue Asp-424, which may play a role in raising the pK of Glu-357 (30), is pseudo-trisaccharide (PTS), glucose, and branched PTSs as modeled maltose molecule. In the context of our proposal, Glu- transglycosylation products (Fig. 4). PTS can be recovered in 332 appears to play an important role in the binding of small pure form from an enzyme reaction mixture. When PTS and glucose, both in large amounts, reacted with ThMA, no trans- ThMA is able to cleave a-D-(1,4)-glycosidic bonds of pullulan, glycosylation product was formed. Therefore, the transglycosy- although it does so inefficiently. It can also cleave a-D-(1,6)- lation reaction by ThMA is concomittant with the hydrolysis of glycosidic bonds of pullulan and small oligosaccharides. The glycosidic bond. Catalysis by a-amylases is widely believed to cleavage is inefficient, and therefore, branched products result- proceed via a double-displacement reaction, in which a covalent ing from the a-D-(1,6)-transglycosylation can be accumulated.
glycosyl-enzyme intermediate is formed and subsequently hy- The extra sugar-binding space, we propose, is also responsible drolyzed (22, 23). Given this mechanism and the fact that the for accommodating branched substrate into the active site cleft transglycosylation reaction requires a high concentration of an for cleavage. It was able to place a short pullulan molecule acceptor sugar, it is reasonable to think that the acceptor containing two maltotriose units into the active site cleft with- molecule competes with a water molecule at the active site for out a severe steric clash so that an a-D-(1,4)-glycosidic bond attacking the glycosyl-enzyme intermediate. The assumption after a maltose unit is positioned close to the catalytic residues.
requires a sugar-binding site adjacent to the main substrate- Clearly, the extra sugar-binding space was necessary in fitting binding site. Consistently, an extra space, lined by both the the molecule.
N-domain and the (a/b) barrel, is found deep at the bottom of the active site cleft (Fig. 5). A corresponding space is not found in the smaller amylases. Restrained substrates, such as CD, MAases, NPases, and CDases are intracellular enzymes, cannot reach the space as the bound b-CD model suggests. The which implicates their biological roles in metabolizing small size of the space appears to accommodate a disaccharide such oligosaccharides, including CDs imported into the cells for en- as maltose rather easily, as a maltose molecule can be success- ergy generation, and storing them in the form of branched fully docked into the space (Fig. 5b). In this model, the C4-OH oligosaccharides under the condition of high concentration of group of a maltose at the nonreducing end is 3.6 Å away from glucose or maltose. A closely linked enzyme, CGTase, is hy- Glu-357. There is some degree of freedom to rotate and trans- pothesized to have evolved from an ancestral hydrolase on the late the model without steric clash. We propose that the space basis of sequence and phylogenetic analysis (26). The conser- (referred as "extra sugar-binding space") is responsible for the vation of the common (a/b) barrel and C-domain in the struc- transglycosylation activity of ThMA. A mono- or disaccharide ture of ThMA also indicates that the three groups of the en- occupying the space could serve as an acceptor molecule to zymes are likely to have evolved from a common ancestor.
compete with a water molecule for attacking enzyme-substrate Probably, these enzymes add a significant adaptive value to intermediate catalyzed by the same catalytic armory as shown organisms by enabling efficient metabolism of starch materi- in Fig. 6. In the extra sugar-binding space, an acceptor sugar molecule may be able to position either the C3-, C4-, or C6-OH Most members belonging to the a-amylase family exhibit the group in a proper orientation for nucleophilic attack of the single activity of hydrolyzing a-D-(1,4)-glycosidic bond. The glycosyl-enzyme intermediate. The idea of extra sugar-binding unique addition of ;130 residues at the N terminus of these space playing an important role in the transglycosylation ex- enzymes compared with the other amylases has been specu- plains the effect of Glu-332 mutation. We found that Glu-332 is lated to be responsible for a rarely found example of an enzyme absolutely conserved among 13 different related enzymes acquiring a new catalytic activity. We proved that ThMA is a known so far, and the corresponding residue in the smaller homodimer in solution. As far as we know, there is no small a-amylases is conserved as histidine (24, 25) (Fig. 1). While the molecular weight a-amylase whose functional unit is a mul- latter is known to be important for substrate-binding, Glu-332 timer. The most salient feature of the dimer is that the unique in ThMA is located at the extra sugar-binding space and does N-terminal domain of one subunit comprises the active site not interact with the bound b-CD model. Substitution of the together with the (a/b) barrel of the other subunit. The active residue by histidine results in the drastic reduction of a-D-(1,6)- site is otherwise similar to those of the smaller a-amylases in transglycosylation products without affecting the hydrolysis that it is located at one end of the (a/b) barrel and contains the activity. The residue is at hydrogen bonding distance from the invariant catalytic residues at similar spatial position. The Crystal Structure of Maltogenic Amylase unique N-terminal domain of ThMA and probably of the re- Carbohydr. Res. 313, 235–246
5. Kim, T.-J., Kim, M.-J., Kim, B.-C., Kim, J.-C., Cheong, T.-K., Kim, J.-W., and lated enzymes plays a major role in the modification of the Park, K.-H. (1999) Appl. Environ. Microbiol. 65, 1644 –1651
common active site structure through the dimer formation for 6. Takata, H., Kuriki, T., Okada, S., Takesada, Y., Iizuka, M., Minamiura, N., acquiring the distinct substrate profile and new catalytic prop- and Imanaka, T. (1992) J. Biol. Chem. 267, 18447–18452
7. Tonozuka, T., Ohtsuka, M., Mogi, S., Sakai, H., Ohta, T., and Sakano, Y. (1993) erty. Both the transglycosylation and the hydrolysis of a-D- Biosci. Biotechnol. Biochem. 57, 395– 401
(1,6)-glycosidic bond by ThMA are explained on the basis of the 8. Oguma, T., Matsuyama, A., Kikuchi, M., and Nakano, E. (1993) Appl. Microbiol. Biotechnol. 39, 197–203
structural analysis and modeling with support from the bio- 9. Kim, T.-J., Shin, J.-H., Oh, J.-H., Kim, M.-J., Lee, S.-B., Ryu, S., Kwon, K., chemical and the mutagenesis studies. It is surprising that the Kim, J.-W., Choi, E.-H., Robyt, J. F., and Park, K.-H. (1998) Arch. Biochem. new catalytic activities are gained not by creating a new cata- Biophys. 353, 221–227
10. Fiedler, G., Pajatsch, M., and Bock, A. (1996) J. Mol. Biol. 256, 279 –291
lytic residue, but most likely by creating the extra sugar-bind- 11. Kadziola, A., Sogaard, M., Svensson, B., and Haser, R. (1998) J. Mol. Biol. 278,
ing space at the active site. Although our explanation needs 12. Fujimoto, Z., Takase, K., Doui, N., Momma, M., Matsumoto, T., and Mizuno, H.
elaboration by structural elucidation of the enzymes in complex (1998) J. Mol. Biol. 277, 393– 407
with different substrates or analogues, the current model may 13. Cho, M.-J., Cha, S.-S., Park, J.-H., Cha, H.-J., Lee, H.-S., Park, K.-H., and Oh, serve as a paradigm for other members of amylases with the B.-H. (1998) Acta Crystallogr. Sec. D 54, 416 – 418
14. Otwinowski, Z., and Minor, W. (1997) in Methods in Enzymology (Carter, activities of transglycosylation and hydrolysis of a-D-(1,4)- and C. W., and Sweet, R. M., eds) Vol. 276, pp. 307–326, Academic Press 15. Navaza, J. (1994) Acta Crystallogr. Sec. A 50, 157–163
16. Bru¨nger, A. T. (1992) X-PLOR, Version 3.843, Yale University Press, New
Finally, the natural design of the transglycosylation activity of the enzyme at high concentration of acceptor sugar mole- 17. CCP4 (1994) Acta Crystallogr. Sec. D 50, 760 –763
cules may now be altered for biotechnological applications to 18. Zamyatnin, A. A. (1984) Annu. Rev. Biophys. Bioeng. 13, 145–165
19. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J.
produce branched oligosaccharides with higher efficiency.
Mol. Biol. 215, 403– 410
20. Kamitori, S., Kondo, S., Okuyama, K., Yokota, T., Shimura, Y., Tonozuka, T., Acknowledgments—We thank Drs. N. Sakabe, N. Watanabe, and M.
and Sakano, Y. (1999) J. Mol. Biol. 287, 907–921
Suzuki for assistance during data collection at Photon Factory (PF).
21. Szejtli, J., August, S., and Richter, M. (1967) Biopolymers 5, 5–10
Helpful advice from Drs. K. K. Kim and H.-S. Hwang is also 22. Koshland, D. E., Jr. (1953) Biol. Rev. 28, 416 – 436
23. Tao, B. Y., Reilly, P. J., and Robyt, J. F. (1989) Biochim. Biophys. Acta 995,
24. Nakajima, R., Imanaka, T., and Aiba, S. (1986) Appl. Microbiol. Biotechnol. 23,
1. Jespersen, H. M., MacGregor, E. A., Henrissat, B., Sierks, M. R., and 25. Svensson, B. (1994) Plant Mol. Biol. 25, 141–157
Svensson, B. (1993) J. Protein Chem. 12, 791– 805
26. del-Rio, G., Morett, E., and Soberon, X. (1997) FEBS Lett. 416, 221–224
2. Kim, I.-C., Cha, J.-H., Kim, J.-R., Jang, S.-Y., Seo, B.-C., Cheong, T.-K., Lee, 27. Adachi, M., Mikami, B., Katsube, T., and Utsumi, S. (1998) J. Biol. Chem. 273,
D.-S., Choi, Y.-D., and Park, K.-H. (1992) J. Biol. Chem. 267, 22108 –22114
3. Cha, H.-J., Yoon, H.-G., Kim, Y.-W., Lee, H.-S., Kim, J.-W., Kweon, K.-S., Oh, 28. Esnouf, R. M. (1997) J. Mol. Graph. Model. 15, 132–134
B.-H., and Park, K.-H. (1998) Eur. J. Biochem. 253, 251–262
29. Honig, B., and Nicholls, A. (1995) Science 268, 1144 –1149
4. Park, K.-H., kim, M.-J., Lee, H.-S., Han, N.-S., Kim, D., and Robyt, J. F. (1998) 30. McCarter, J. D., and Withers, S. G. (1994) Curr. Opin. Struct. Biol. 4, 885– 892

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Future horticulture - case study worksheets

growingfutures case study series 18. Health Enhancing Products from New Zealand plants Our developement of health enhancing products have three important factors: 1. New Zealand's reputation continues to grow internationally as a source of clean and green raw materials, including customised high-tech ingredients that are sold to nutritional manufacturers around the world.


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