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THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 11, Issue of March 14, pp. 9875–9884, 2003 Printed in U.S.A.

Differential Regulation of a Hyperthermophilic Novel (Ca,Zn) Two-metal Center by Zinc*

-Amylase with a

Received for publication, November 6, 2002, and in revised form, December 11, 2002 Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211339200

Anni Linden‡, Olga Mayans‡§, Wolfram Meyer-Klaucke‡, Garabed Antranikian¶, and Matthias Wilmanns‡
From the ‡European Molecular Biology Laboratory, Notkestrasse 85, D-22603 Hamburg, Germany and the ¶Department of Technical Microbiology, Technical University Hamburg-Harburg, Kasernenstrasse 12, D-21073 Hamburg, Germany

The crystal structure of the -amylase from the hyperthermophilic archaeon Pyrococcus woesei was solved in the presence of three inhibitors: acarbose, Tris, and zinc. In the absence of exogenous metals, this -amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a novel, activating, twometal (Ca,Zn)-binding site and a second inhibitory zincbinding site that is found in the 1 sugar-binding pocket within the active site. The data resolve the apparent paradox between the zinc requirement for catalytic activity and its strong inhibitory effect when added in molar excess. They provide a rationale as to why this -amylase, in contrast to commercially available -amylases, does not require the addition of metal ions for full catalytic activity, suggesting it as an ideal target to maximize the efficiency of industrial processes like liquefaction of starch.

-Amylases ( -1,4-D-glucan 4-glucanohydrolase, EC 3.2.1.1) are endo-acting hydrolases that randomly cleave the -1,4glycosidic linkages of branched and linear carbohydrates such as amylopectin and amylose in starch and glycogen (1–3). Their widespread occurrence in various organisms and the consumption of their substrates for food reserves and energy sources have led to intense interest in their biomedical properties and to major biotechnological applications in industry (4 – 6). Their value in specific industrial processes depends critically on their pH and optimal temperature, which vary depending on the organism of origin. Nearly all -amylases belong to class-13 of glycosylhydrolases by virtue of their characteristic sequence motifs. However, in general, their overall sequence similarity is low, which, for some members, falls below the 10% level (1–3, 7). A number of crystal structures of class-13 members have been solved, comprising -amylases from organisms inhabiting environments that span a large temperature range, from psychrophilic to hyperthermophilic (3). These -amylases share a common three-domain fold with the catalytic activity located on the C-terminal face of a central ( )8-barrel, domain A. The
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 1MWO, 1MXD, and 1MXG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http:/ /www.rcsb.org/). § Present address: Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland. To whom correspondence should be addressed: EMBL, Hamburg Outstation, c/o DESY, Notkestr. 85, D-22603 Hamburg, Germany. Tel.: 49-40-89902-126; Fax: 49-40-89902-149; E-mail: wilmanns@ embl-hamburg.de.
This paper is available on line at http://www.jbc.org

sequences of the other two domains, B and C, are void of any conserved motifs and are not involved directly in substrate catalysis. Besides the recently solved three-dimensional structure of the glycosyltrehalose trehalosidase from Sulfolobus solfataricus Km1 that exhibits -amylase activity (8), no other structures of archael class-13 -amylases are known to date. The -amylase from the hyperthermophilic archaeon Pyrococcus woesei (PWA),1 which is identical to that from Pyrococcus furiosus (9), was cloned and classified as a class-13 glycosylhydrolase (10). Throughout this study, both the -amylases of P. woesei and P. furiosus will be referred to as PWA. Biochemical characterization of recombinant PWA revealed a high thermal stability and maximum catalytic activity at 100 °C (10 – 12). In a recent study using coupled plasma-atomic emission spectroscopy, it was shown that PWA binds calcium and zinc stoichiometrically and with high affinity (13). Thus, in contrast to many other -amylases, PWA activity does not require the addition of metal ions. Due to its superior properties, exceeding those of -amylases currently employed in commercial preparations (5, 14), PWA has become a prime candidate for maximizing the efficiency of applications in the starch industry. To reveal the molecular basis of its properties, we solved the x-ray structure of PWA in the presence of three different active site ligands. The structure reveals a novel (Ca,Zn)-binding site in close proximity to the active site cleft, which is not found in -amylases of any bacteria, plants, and most other Archaea. The presence of this site indicates adaptive evolution of PWA to the specific living conditions of P. woesei. The three complex structures also show how competitive binding of organic compounds and zinc provides a direct molecular explanation as to why a molar excess of zinc or some chemically related metals inhibits PWA activity.
EXPERIMENTAL PROCEDURES

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Gene Amplification and Expression—The wild-type gene encoding PWA was amplified by PCR using primers deduced from the open reading frame of the P. furiosus wild-type -amylase gene (10). An NcoI recognition site (underlined) was fused to the 5 -end of the sense primer (5 -CAT GCC ATG GAC ATA AAG AAA TTA ACA CCC CTC-3 ), as was an XhoI recognition site (underlined) to the antisense primer (5 -GGC CTC GAG TCA CCC AAC ACC ACA ATA ACT CCA-3 ). Additionally, the natural GTG start codon was replaced with the Escherichia coli start codon ATG (boldface). The PCR product was digested twice with NcoI/XhoI and cloned into the NcoI/XhoI cloning site of the pET-15b vector (Novagen, Madison, WI) to generate the plasmid pPWA. pPWA was cloned and expressed in E. coli strain BL21(DE3) (15) as described previously (12). The deduced amino acid sequence is identical to the

1 The abbreviations used are: PWA, P. woesei -amylase; PIXE, proton-induced x-ray emission; MES, 4-morpholineethanesulfonic acid; Ac, acarbose; BLA, B. licheniformis -amylase.

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the central domain A.59 keV ( 1. For PWA Ac/Zn.12 M Britton and Robinson buffer (pH 12) and purified by hydrophobic interaction chromatography using a phenyl-Superose HR 5/5 column (Amersham Biosciences. domain B of PWA is one of the smallest -amylase B domains. and acarbose and Tris molecules were built manually using the graphics software program O. CA) and shock-frozen in a nitrogen stream at 100 K. PWA Tris) and 1196 reflections (1. 2) the specific substrate analog acarbose and zinc (PWA Ac/Zn). Comparison of the overall structure of PWA with that of other known -amylases using the program DALI (26) revealed the highest structural similarity to the homologous enzymes from the hyperthermophile B. As in other -amylases. Metal atoms were identified from high peaks in the difference Fourier maps and in part from additional high peaks in the anomalous difference Fourier maps. The measurements were calibrated using the nine sulfur atoms of the PWA sequence (five cysteines and four methionines) as an internal standard.1%. 2.2) and concentrated to a final concentration of 5. The initial R-value was 44. 3). is folded as a ( )8-barrel and contains the active site at its C-terminal face. no transfer into a cryoprotectant was carried out. which is above the k-adsorption edge of zinc of 9. Metals were refined as calcium when no higher peak level appeared in the anomalous electron density map and the coordination geometry agreed with that of known protein calcium complexes. A heavy atom derivative suitable for crystallographic phase determination was obtained by soaking PWA Zn in a solution containing 29% (v/v) polyethylene glycol monomethyl ether 550 and 1.1 M sodium cacodylate (pH 6. Crystallization—Purified recombinant PWA was crystallized at a concentration of 5. root mean square deviation of 1.6 and 80. 40% (v/v) ethanol and 31.7%.5% (v/v) polyethylene glycol 400 were used as a cryoprotectant.02 M triethanolamine containing 30% (v/v) isopropyl alcohol (pH 7.1 M MES (pH 6. respectively. Overall Structure and Ligand Composition—We have solved the structure of PWA in three different forms using experimental phases from a mercury derivative. A total number of 25. 4). Eschborn. initial phases were calculated and refined using SHARP (20) up to 2. resulting in a single solution with correlation coefficients of 68.0 mM CH3HgCl for 1.1 M Tris (pH 8. and 3) Tris.5-Å resolution. and 1 l of 0. Inclusion bodies were solubilized in 0.5) containing 0. and PWA Tris complexes were recorded up to 2.2-. 29% sequence identity. Weizheng. RESULTS -amylase gene from P. Further data statistics are presented in Table I.5 mg/ml by the hanging drop vapor diffusion method. The samples ( 2 l. 2. and Y. PWA Ac/ Zn) for the calculation of Rfree was excluded during refinement in CNS. forming a small antiparallel -sheet and a short 310-helix (Figs. respectively. covering residues 1–109 and 170 –340. The protein was eluted in 0. Its secondary structure is limited to two short -strands. The model was traced with the help of Bacillus licheniformis -amylase (BLA) as a template.5 h at 19 °C prior to x-ray data collection. Heavy atom positions were determined from mercury derivative difference Patterson maps using the RSPS program.967 reflections in the resolution range of 20 –2. and 1. Solvent building and subsequent refinement were performed as described above. which would otherwise have changed the x-ray signal intensity.17 coulomb was applied within 20 min. They have been refined to 2. 1 and 2). Metals were refined as zinc when the crystal growth conditions contained zinc ions and if the metals sites showed a high anomalous peak emerging from data set collection at 12.org by guest.5 Å. root mean square deviation of 2.02 M triethanolamine (pH 7. unpublished data. The structures of the two PWA inhibitor complexes (PWA Tris and PWA Ac/Zn) were solved by molecular replacement using the structure of PWA Zn as a search model. thus forming part of the active site cleft. and 1.5-Å resolution. J. PWA Zn was grown from 1 l of protein solution and 2 l of 0. An initial map calculated in CNS (21) was used to build the model of the molecule using the interactive graphics program O (22). 1). 2010 2 C. and Reduction—Each x-ray data collection was performed with one single crystal using 27% (v/v) polyethylene glycol monomethyl ether 550 as a cryoprotectant for PWA Zn. which was used as a buffer for crystallization trials in the absence of zinc (PWA Tris). Yunyan.9 m polyethylene terephthalate foil. 2). and the characteristic x-rays were detected using a germanium detector. licheniformis (BLA. ensuring that no elemental loss (sulfur or metals) due to thermal stress occurred.5-Å resolution was collected from a PWA mercury derivative isoform at a wavelength above the L-III adsorption edge of mercury.05 M Zn(OAc)2 and 35% (v/v) 2-methyl-2.5) containing 0.4-pentanediol at 25 °C. Germany). Fractions containing amylolytic activity at 99 °C were pooled and further purified by size-exclusion chromatography using a Superdex 200 16/60 prepgrade column.4 keV ( 0. Covering a range of 58 residues only. unless stated otherwise. X-ray Data Collection.05 M MgCl2 and 40% (v/v) ethanol. Initial phases for PWA Zn were obtained using single isomorphous replacement including the one-wavelength anomalous scattering contribution (SIRAS).0-.7%. A disulfide bridge is formed by two consecutive cysteine residues (Cys153 and Cys154) in close vicinity to the zinc-binding site of this two-metal center. Rotation and translation functions were calculated with AMoRe (25) using x-ray data from 10 to 2. The concentration of the enzyme preparation was determined by estimating the extinction coefficient according to the method of Gill and von Hippel (16). PWA Ac/Zn.28 Å).5% for PWA Tris and PWA Ac/Zn. all elements besides those lighter than sodium can be detected in a single scan at a minimum weight limit of 1–10 ppm. Germany). furiosus (10) and to the P. and Model Evaluation—Programs used for the subsequent calculations were from the CCP4 program suite (19). Structure Determination. The structures are in the presence of three different inhibitors: 1) zinc (PWA Zn).2 Purification and Concentration Determination—The recombinant -amylase gene product was purified from catalytically active inclusion bodies (12). merged. Protein samples were dialyzed extensively against Chelex 100-treated buffer composed of 10 mM sodium phosphate (pH 12) to remove chloride and sulfur compounds that disturb the protein sulfur signal. X-ray data sets for the PWA Zn. Phases were improved by solvent flattening with Downloaded from www. An anomalous diffraction data set to 2. The data were processed. Initial phases of the PWA Tris model were applied to automated model building using ArpWArp for model completion. solvent building was performed using the solvent 0 mode of ArpWArp (24) and CNS with waters placed in local maxima of difference electron density maps above 3 . domains A and B comprise a novel (Ca.9876 Structure of a (Ca. The final model was built in a consecutive cycle of crystallographic refinement using CNS and manual rebuilding.5 mg/ml in an Amicon ultrafiltration device (Millipore Corp. Among those with a known structure. 1 mg/ml) were dropped onto sample holders covered with a 0. which is described further below (see Fig. Riverside. The models were verified and corrected. respectively. Metal Analysis—The metal content of isolated PWA was determined by detection of the proton-induced x-ray emission (PIXE) at the Leipzig 2 MeV proton microprobe (17). The amylase was eluted in 0.8% (995 reflections) set aside for cross-validation (23). At their interface. woesei amylase sequence released to the NCBI Protein Database by Lu et al. with 3. PWA displays the canonical glycosylhydrolase class-13 fold that is composed of three domains.2-. Processing. Freiburg.91 Å). Domain B (residues 111–169) inserts between -strand 3 and -helix 3a of domain A.0-. Lu. A test set of 1686 reflections (4. A sample area of 1 mm2 was scanned with the proton microbeam. whereas other members of the family span 100 residues (2. From three major binding sites. They were refined as magnesium sites when an excess of magnesium was present during crystallization and a negative Fo Fc difference Fourier electron density peak appeared when the metal position was refined as calcium.5) containing 0. and the free Rfactor was 42.Zn) metal center.2).01 M Zn2(SO4) 7H2O and 25% (v/v) polyethylene glycol monomethyl ether 550 at 19 °C.2%.1% and R-factors of 39. on November 14.. and 1 l of 0. 1 l of protein solution.8 Å) (Fig. The PWA Ac/Zn complex was grown from 1 l of acarbose solution (11 mg/ml). domain B of BLA is most .22 Å were included in the refinement. For PWA Tris. The number of metals per molecule was calculated from their calibrated signals with reference to the calibrated sulfur signal.6-Å resolution.Zn) Metal Center-containing -Amylase SHARP. A total charge of 0.4 and 33. and scaled using the HKL program suite (18). 1 l of protein solution. Using this technique. After several cycles of positional and restrained individual B-factor refinement.jbc.5 Å based on comparison of the C backbone trace) and from barley (28% sequence identity. The PWA Tris complex was crystallized from 1 l of acarbose solution (11 mg/ml). The overall accuracy was estimated by taking into account the statistical errors of the x-ray yield and the uncertainty in the correction for x-ray absorption within the sample. Refinement. A–C (Fig. All crystals were mounted onto nylon cryo-loops (Hampton Research.

PWA was expressed heterologously in E. Three zincbinding sites (Zn3. The presence of an anomalous signal under the experimental conditions of x-ray data collection was used to indicate the presence of zinc (Tables I and II). Downloaded from www. A. top view of the PWA Ac/Zn complex. domain B of BLA contains 43 additional residues that form a second -sheet (27). the type of the metal sites was characterized by the analysis of anomalous x-ray data and positive peaks in Fo Fc difference Fourier electron density maps (Table I). Domains A–C are colored cyan. 1.6dideoxy. These metal . The remaining four sites. The two inhibitors are superimposed onto each other.org by guest. In contrast. and brown. without an anomalous signal.1 0. C.4 molar eq of zinc and 4 2 molar eq of calcium bound to PWA. and nitrogen atoms are blue. and Mg6). when refined as an ordered solvent ( 10 ). all sites except one showed a significant anomalous signal and were therefore refined as zinc sites. all three structures have five metal-binding sites in common (Table II). PWA Tris shown from the same orientation as in A. which is virtually identical to the nitrogen position of the 4-amino-4. Thus. and magnesium and calcium. In the PWA Tris structure. bringing the total in these structures to seven. respectively.Zn) Metal Center-containing -Amylase 9877 FIG. The PIXE data revealed the presence of 1. Zn5. magenta. The two crystal forms grown in the presence of zinc (PWA Zn and PWA Ac/Zn) reveal two additional metal sites. green. the PWA structures show excessive metal binding. and Zn6) of PWA Ac/Zn are replaced by magnesium ions in PWA Tris (Mg3. In contrast. The C-terminal domain C (residues 341– 435) is arranged in an eight-stranded antiparallel -sheet containing a Greek key motif. were interpreted to be calcium or magnesium sites. oxygen atoms are red. coli and purified in the absence of exogenous metals except for sodium. in the PWA Zn and PWA Ac/Zn structures. Based on these data. the -strands composing the -barrel of domain A are colored gray. solved from crystals grown in the absence of zinc. respectively. We initially identified the nature of the bound metal ions by PIXE. orange) and acarbose molecules are indicated and numbered according to Table II. 2010 similar (root mean square deviation of 2. but retaining strong positive difference electron density. The remaining site was interpreted as a calcium site. Overall crystal structure of PWA complexed with different ligands. PWA Ac/Zn rotated 90° along a horizontal axis with respect to the orientation in A. The cylinders and arrows represent helices and -strands. Bound metals (zinc. For these experiments. In PWA Tris. For clarity. but in the presence of magnesium. the stoichiometry of calcium (or magnesium) and zinc sites observed in the PWA Tris structure is in good agreement with the PIXE data.jbc. on November 14. only one site showed a significant anomalous signal and therefore was refined as a zinc site (Table II). Mg5. the first of which is acarbose and the second of which is a coordinated zinc ion. The backbone of the acarbose molecules is yellow.Structure of a (Ca. no metal is found in site 4 of the PWA Ac/Zn structure. In the PWA crystal structures.3 Å based on the C backbone) (27) to the corresponding domain of PWA.-D-glucose ring of acarbose. B. The active site cleft at the front face of the PWA molecule contains two inhibitors with partial occupancies. The function of this domain still remains unclear.

2. which is fused to maltose. one acarbose molecule is . sites will be referred to as activating sites (Table II. 3). including commercially available BLA (10. we also assessed the regulatory properties of a number of metal ions (Table III). AVA. sites 1 and 2). Based on the measured inhibition data. zinc had the strongest inhibitory effect. those coordinating calcium (site 2. including the transition state analog acarbose and the buffer Tris (Table III). Acarbose is a pseudotetrasaccharide inhibitor consisting of a valienamine unit at the nonreducing end linked to 4-amino4. Table II) in domain B are colored yellow. Structure-based alignment of PWA sequences.-D-glucose. Table II) are highlighted in red. In the PWA Ac/Zn structure. In contrast to many other characterized -amylases. and other sites (sites 3– 6). Chemically Unrelated Inhibitors Competitively Bind to the Active Site—We quantified the inhibitory effects of a number of established -amylase active site ligands.org by guest. Hordeum vulgare -amylase (AMY2) chain A (Protein Data Bank code 1AVA) (46). The secondary structure assignments for PWA are shown as colored cylinders (310-helices and -helices) and arrows ( -strands) above the aligned sequences. 2010 FIG. 13). resulting from co-crystallization of PWA in the presence of acarbose and zinc. we selected three ligands (acarbose. an inhibiting site (site 7). on November 14. and zinc) for crystallographic characterization of the PWA active site (Fig.9878 Structure of a (Ca.Zn) Metal Center-containing -Amylase Downloaded from www. cf. The seven invariant amino acid residues are marked in black. The positions of amino acid residues coordinating the zinc ion in domain B (site 1. To link the observed tight binding of calcium and zinc to a potential role in activity regulation. PWA was not activated significantly by an excess of calcium (Table III). cf.jbc. residual PWA activity was 10%. The alignment was carried out with DALI using the main chain positions of each coordinate set. In the presence of 3 mM zinc. The Protein Data Bank code for BLA is 1BLI (27).6-dideoxy. and the cysteines forming disulfide bonds are colored green. Except for copper (full inhibition in the presence of 3 mM Cu2 ). Tris.

3 19.c Completenessb Refinement Refinement range (Å) R factor (%)d Rfree (%) No.6-dideoxy.0 19. a Tris molecule entirely replaced acarbose in the active site of PWA (PWA Tris). However.2 106.4 21.9 (6.39 20. 2 and 3.4 3975 3570 398 7 0.02–1. like the amino group of the 4-amino-4. space group P212121 a b c No.22 2.005 1.5 76.410 3.007 1.22 62. 2010 All beamlines are at the DORIS storage ring at EMBL/DESY (Hamburg.9 25.4.7 (93.67 99.8 30. However. 34 –36) and demonstrating its function as a potent competitive inhibitor (34. acarbose and zinc. 3. Soaking of the PWA Zn crystal form in a solution containing 15 mM acarbose at pH 6. C and D).1) 4. is bound by the carboxylate group of Glu222.-D-glucose (Glu222 and Asp289) groups of acarbose in the PWA Ac/Zn (Fig.6 35.2 106. Therefore.9102/13.d.8) 97. In the third crystal form (PWA Zn). bound to the active site at the C-terminal face of the ( )8barrel of domain A. if acarbose was added in 100 mM Tris buffer without zinc.302 6.51–2. confirming previous structural data from a number of -amylase Tris complexes (32.4 26. indicating that PWA is not catalytically active under crystallization conditions.6dideoxy.8 (3. I is the observed intensity.5 75.2 19.-Dglucose group of acarbose (Fig.45 2. 3.5 (32.5 14.0 (20.4 (88. are visible.s. of non-hydrogen atoms/asymmetric unit Total Protein Solvent Other ligands r. Comparison of the PWA Zn and PWA Ac/Zn structures reveals that the position of the zinc ion in PWA Zn is nearly identical to that of the acarbose nitrogen and the partially occupied zinc ion in the PWA Ac/Zn complex.006 1. 38).9 17.28–2. zinc binds to the carboxylate group of the same residue (Glu222) (Fig. E and F. Tris is bound by residues that interact with the valienamine (Arg196. The occupancies of the two inhibitors.5 39. A and B.-D-glucose ring of acarbose. The location and orientation of the acarbose inhibitor within the active site of PWA resemble previous data from several -amylase acarbose complexes. A and B).3 BW7B 0. 4C). A and B) and Tris (C and D). where h represents a unique reflection and j is symmetry equivalent indices.9) 6.m. 3.0) 20–1. were refined to final values of 0. entirely inhibits the amylolytic activity of PWA.4 BW7A 1.97 2.6dideoxy.9 78.0–2. Germany).6 and 0. comprising the acarviosine residue and a linked glucose ring. 3.8 (69.55–1. We noticed that residual anomalous difference electron density remained at the nitrogen position of the 4-amino-4. Three of its four sugar rings (A–C). 5. His288.0 22. On the other hand. a zinc ion was placed into this position as an alternative inhibitor (Table II.8 31.4 10. We antic- .27° 15. in contrast to previous observations (29 – 33). We reasoned that the low temperature used for crystal growth of this hyperthermophilic -amylase has been sufficient to inhibit any transglycolytic catalysis within the crystal. at concentrations 5 mM. Numbers in parentheses refer to the highest resolution shell of observed data.3 26.3 (87.8453/14.4) BW7B 0.292 8.32° 23. Thus.0) 94. but the forth unit accounting for the last glucose unit at the reducing end of the molecule is not. d Rfactor Fc(h) / h Fo(h) .5 18. and Table II.8) 7.9 4007 3574 289 144 0. Two additional solvent molecules are located at O-4 and O-6 of the acarbose complex and one near O-3. PWA does not display acarbose transglycosylation activity. respectively.8 77. The carboxylate group of Asp289 interacts with the zinc ion by a solvent-mediated hydrogen bond.4) 96.1) 20–2. A and B) (2. where Fo and Fc are the observed and calculated structure factors.Structure of a (Ca. of unique reflections Multiplicity I / (I) b Rsym (%)b.2 4252 3574 509 169 0.e from target values Bonds (Å) Angles Average B-factors (Å2) Main chain Side chain All atoms a b c X11 0.1) 20–2.4 16.8453/14.0) 6.7 (4.0 (17. a molecular rationale for previous observations (10) and our data (Table III) showing how zinc.52 51. h Fo(h) e Root mean square deviation. The structural overlay of acarbose and zinc within the active site of the PWA Ac/Zn structure displays directly the competitive nature of these two chemically unrelated PWA inhibitors (Figs.5 confirmed partial replacement of zinc by the inhibitor (data not shown).45 62.5 (21. respectively.org by guest.97 62.6 15.0–1. 37.52 1.2 (3. thus substituting the oxygen atoms of the glucose ring of the natural substrate bound to the 1 subsite.004 3.9 78. the PWA Zn structure permits a more detailed description of the coordination geometry of the zinc ion.67 99. 3.3 18. Rsym Ih / h j Ihj . 28). and 4C).Zn) Metal Center-containing -Amylase TABLE I X-ray data and structure refinement statistics PWA Zn PWA Zn (Hg soak) PWA Tris 9879 PWA Ac/Zn Data collection Beamlinea Wavelength (Å)/energy (keV)b Resolution range (Å) Highest resolution shell (Å) Unit cell dimensions (Å). on November 14. for the first time.0008/12. site 7) that binds to the 4-amino-4. and h j Ihj I is the mean value of I.5 136.-D-glucose ring within the active site of the refined PWA Ac/Zn complex (Fig.2 106. and 7 of the ( )8-barrel with the subsequent helices (Figs.6-dideoxy.0–2.jbc. Zinc. site 7).0–1.34° 29.2 Downloaded from www.9) 89. and Asp289) and 4-amino-4. Two solvent molecules are located at positions equivalent to C-7 and O-2 of the valienamine residue in the PWA Ac/Zn complex (Fig.62 20. the PWA Zn structure provides. 3. It interacts with a number of highly conserved residues that reside in loops connecting -strands 4.

Each reaction was carried out with 11. Ile292 Zn Zn 24. Cys166 Zn Zn 27. The zinc ion is coordinated by ligands in a distorted tetrahedral geometry.2 24.Zn) Metal Center-containing -Amylase observations that magnesium does not inhibit enzyme activity (10. the number of protein ligands in this calcium-binding site exceeds those found for the same site in other class-13 -amylases (2). the catalytic activity of PWA at high temperatures is dramatically reduced. three other acarbose sites have been identified in the two structures in which acarbose was present in the crystallization medium (PWA Ac/Zn and PWA Tris) (Fig. Thus. Relative activity Additives 1 mM 3 mM % 5 mM Mg2 Ni2 Ca2 Fe3 Co2 Fe2 Mn2 Zn2 Cu2 EDTA Tris Acarbose 106 60 105 95 92 85 58 25 5 95 91 74 106 51 94 82 66 55 44 7 0 91 87 54 99 86 83 63 55 38 33 0 0 86 83 20 ipate that the strong inhibition of many -amylases by zinc (39) is via the same binding to the 1 site as observed in PWA Zn.0/9.1 14. Generally. b Glu36. Glu253 Zn 37.7 Lys33.9 34. In contrast.2/9.9 16. In the PWA structure.” The values for the anomalous peaks (ano) were obtained from the anomalous difference electron density maps calculated using fast Fourier transformation.4 12.5 12.2 14. 2010 Mg 11. it is most likely that the observed metal ions originate from the medium that was used for heterologous expression of PWA in E. Gly157 .4 Å (Fig. class-13 glucosidases contain a common calciumbinding site (40). Asp164.0/59. 41). Therefore.2/– c No metal Main chain oxygen involved in metal coordination. In contrast to the first metal site (Table II. The third acarbose-binding site (Ac-III) is located at the surface of domain B at a distance of 30 Å from the active site (Fig.8 40. The measurements were performed according to Bernfeld (51) in 0. His152. Thus.9/– Asn110.05 M sodium acetate and 1% (w/v) starch (pH 5.0 Mg 18.3–7.9 27. Glu350 Zn Zn 24.5 ng of PWA for 4 min at 94 °C. Asp155. including the imidazole groups of His147 and His152. 4A and Table II. Gly157.5 12.1 His147.7 15. Gly202 from a 310helix between -strand 4 and -helix 4 of domain A. 4A). there is a strong anomalous contribution at the second site (site 2). on November 14.0 11.9 15.1 24. The second acarbose-binding site.0/15. at present.9880 Structure of a (Ca. c Symmetry-related intermolecular contact.2 Zn 12.0 14.5/– 25. 1). Side chain residue coordinating zinc observed only in PWA Zn. and the loop connecting -helix 8b of domain A and the first -strand of domain C (Fig.4/14.4/– Mg 12. thus indicating a hetero-population of metals at this site.8/– No metal Downloaded from www. confirming it as common calcium site. Two Different Metals Bind Near the Active Site—All three PWA structures contain a two-metal center in close proximity to the active site cleft. d Main chain carbonyl oxygen involved in metal coordination observed only in PWA Tris. thereby abolishing the zinc site of this two-metal center. coli. to stabilize a catalytically active conformation in PWA at high temperatures.8/9.a. Therefore. A similar carbohydratebinding site was reported for a chimeric Bacillus amyloliquefaciens/licheniformis -amylase (32). this site is matched by a peak without an anomalous signal.3 12.5 9.9/8. Asp349.8/– Asp347.2/11. This has been identified as a zinc site because no other metal with a measurable anomalous signal under the conditions of x-ray data collection was found in the PIXE analysis. with three acarbose rings visible (Ac-II). three residues from the loop preceding -helix 3a of domain A (Asp155 as bidendate ligand. 43). confirming previous biochemical . site 1). the PWA structures do not indicate that magnesium binds to the active site. c Asp252.2/11. Only one of the two sites showed an anomalous signal under the energy conditions used for x-ray data collection (Table I).8 27. The calcium ion is coordinated by seven protein ligands in a distorted octahedral geometry involving the conserved residue Asn110 from loop 3 (domain A)– 1 (domain B). both sites of this two-metal center have a common role. Glu80. Lys323 Zn Zn 19. This site corresponds to the so-called accessory carbohydrate site reported for the pig pancreas -amylase structure (44).org by guest.7/– 15. the sulfhydryl group of Cys166. However.9 10. 1). only a brief account of these sites is given. TABLE III Effect of metal ions and chemical reagents on PWA activity The relative activity refers to the enzyme activity determined without any additive (100 %). Glu88. Gly202a Zn Zn 26. which is essential for their catalytic activity (2. Asp256. This coordination geometry is typical for protein zinc complexes (42. and one ordered solvent molecule (Fig. confirming previous data (13). and Asp164). 11).7 16. irrespective of specific crystallization conditions and ligand binding to the active site.7 41. indicating loss of thermostability (13).1/9. Additional Ligand-binding Sites—The high resolution data of the three PWA structures have allowed the identification of additional ligand-binding sites (Table II).9 13.4 Gly24. 1).1/10. If the cysteine ligand (Cys166) is replaced. and an ordered solvent molecule.jbc. The two metal sites are separated by 7.2 Glu87. Apart from the acarbose site within the PWA active site that is observed only in the PWA Ac/Zn structure. sites 1 and 2) and are located within the interface of domains A and B. is located in a slight depression formed by the 310-helix preceding -strand 1 of domain A.7/5.9 Glu222 Zn 36.2 16. it remains largely unknown as to whether and to what extent these additional sites are critical for thermal stability and catalytic function of the enzyme.5). The fourth acar- TABLE II Metal-binding sites in PWA of different crystal forms The values for the levels of the metal sites were obtained after determination of the positive difference peaks (diff) in the Fo Fc difference Fourier electron density maps as described under “Experimental Procedures. PWA Zn PWA Ac/Zn PWA Tris Site 1 Metal B-factor (Å2) (diff/ano) Protein ligands: Site 2 Metal B-factor (Å2) (diff/ano) Protein ligands: Site 3 Metal B-factor (Å2) (diff/ano) Protein ligands: Site 4 Metal B-factor (Å2) (diff/ano) Protein ligands: Site 5 Metal B-factor (Å2) (diff/ano) Protein ligands: Site 6 Metal B-factor (Å2) (diff/ano) Protein ligands: Site 7 Metal B-factor (Å2) (diff/ano) Protein ligands: a b Ca Ca Ca 27. the loop connecting -helix 6b and -strand 7 of domain A.d.9 35. Glu318.

2. D. Phe159. site 3) at the loop connecting -strands 1 and 2 of domain C (Fig. and E.Structure of a (Ca. left panel). and the C terminus of the protein molecule. 2010 FIG. All three PWA structures display an additional metal-binding site (Table II. A. sites 3– 6) depends on the presence of zinc during crystallization. Hydrogen bonds are shown by dashed lines. Tris (PWA Tris.5 . bose-binding site (Ac-IV) is located at the surface of the domain A/C interface (Fig. respectively. zinc) is contoured at 2. These three rings interact with residues from -helix 8b of domain A and -strand 5. C. 1).Zn) Metal Center-containing -Amylase 9881 Downloaded from www. In E. A and B). the following loop of domain C.Zn) metal center. Zinc ions and solvent molecules are shown as green and gray spheres.0. structures of active site residues in the presence of bound ligands. Active site of PWA with bound acarbose and zinc (PWA Ac/Zn. respectively. C and D). Solvent molecules mediating protein-inhibitor interactions are indicated in B. a zinc ion was modeled in this site. and Tyr199 are not shown. and F.org by guest. on November 14. 4B. Tris. which is coordinated by the three carboxylate . acarbose. schematic representations of the ligands bound to the active site. for clarity. the anomalous difference peak (red) is contoured at 3.0. and 4.jbc. and E. Tyr62. C. and zinc (PWA Zn. B. Each Fo Fc difference electron density map (green) in the absence of ligands (A. 3.7 . again with three sugar rings visible. where the presence/absence of the anomalous difference does not depend on crystallization conditions. In the PWA Ac/Zn and PWA Zn structures. The presence of an anomalous difference at the remaining metal-binding sites (Table II. D. suggesting that metal binding at these sites is weaker and less selective than at the (Ca. and F. E and F).

site 3). A. Asp349. Table II. 3 and contoured at 4. In the PWA Ac/Zn structure. The residual anomalous difference electron density at the zinc position is shown at a contour level of 3. Right panel. In PWA. sites 5 and 6). The structure suggests that this metalbinding site generally serves a stabilizing role.6-dideoxy. the site bears a magnesium (or calcium) ion that is coordinated by the same residue ligands and three ordered solvent molecules in a distorted octahedral geometry (Fig. but also serves as an inhibitory metal-binding site of limited specificity. Two other metal-binding sites within the interfaces of symmetry-related molecules have been found in the PWA structures (Fig. The two chemically related metals Cu2 and Zn2 show comparable inhibition of PWA catalysis. 1 and Table II. structure of the two-metal (Ca. and Glu350 in a trigonal planar geometry. involving at least one lysine residue as ligand. in contrast. this site not only binds organic compounds like acarbose and Tris. two additional solvent molecules lead to a different overall coordination geometry. Asp349. Left panel. However. Asp155. Unique metal-binding sites in PWA. which is superimposed on the bound acarbose ligand. 2010 groups of Asp347. sites 1 and 2).9882 Structure of a (Ca. in the PWA Tris structure. which is a rare feature in known protein crystal structures (43). B. DISCUSSION Zinc Is a Competitive Active Site Inhibitor That Binds to the Conserved 1 Active Site Pocket—Acarbose and related sugar units containing -amylase inhibitors bind to at least three specific active site pockets in class-13 -amylases. One additional metal-binding site in domain A (Table II. The Fo Fc difference and anomalous difference maps are colored as described in the legend to Fig.org by guest.Zn) Metal Center-containing -Amylase FIG. and an ordered solvent molecule (red). structure of the variable PWA metal-binding site in domain C (cf.Zn)-binding site in PWA (cf.2 .jbc. suggesting that they indeed bind into the same pocket and that metal inhibition may correlate generally with binding into the 1 active site pocket (Table III). Although the protonation state of the “metal” site in the organic inhibitors (the ternary amino group in Tris and the secondary amino group of the 4-amino-4. a magnesium ion is coordinated by the same side chain residues as zinc in the left panel. 2) and a solvent molecule. In the PWA Tris structure. C. the total number of metals coordinated by a single PWA -amylase molecule exceeds the number of bound metals previously reported for any other class-13 glycosylhydrolase. right panel). and Glu350) and a solvent molecule (red sphere). The three PWA structures demonstrate that the 1 pocket of the enzyme is the key site for competitive binding by the unrelated inhibitors used in this study. this site is also liganded by an ordered solvent molecule. and Gly202 (cf. respectively. Downloaded from www. Asp164. Fig. The zinc ion (gray sphere) is coordinated by two histidines (His147 and His152). 4B. zinc (green sphere) is coordinated by three negatively charged amino acid side chains (Asp347. one cysteine (Cys166). Overall. The observed high number of bound metals on the protein surface may enhance the hyperthermostability of PWA and may reflect one strategy of evolutionary adaptation to a high temperature environment.5 and 33 . shown is the structure of the partially occupied inhibitory zinc-binding site in the 1 active site pocket of PWA. The calcium ion is coordinated by the conserved residues Asn110. Table II. 4. on November 14. Gly157. site 4) is found only in the presence of zinc.-D-glucose . which may be further enhanced by a nearby disulfide bridge connecting Cys388 and Cys432.

we have solved the PWA crystal structures and revealed the molecular basis for tight calcium and zinc binding by the identification of a nonconserved and PWA-specific (Ca. Two of these proteins are members of the alcohol dehydrogenase family. the addition of calcium inhibits glucoamylases and destabilizes glucose isomerases. which shares the highly conserved calcium-binding site and displays an additional calcium-binding site at a distance of 7 Å (45. The atypical disulfide bridge may stabilize the non-planar semiquinone form of the enzyme’s prosthetic group pyrroloquinoline quinone (49). Interestingly. PWA has been proposed as an alternative to BLA to further improve the efficiency of industrial processes in starch liquefaction (5. chemical. However. 49) and ethanol dehydrogenase from Pseudomonas aeruginosa (50). As such. such as the liquefaction of starch. including those of P.jbc. The 1 pocket residue ligands are highly conserved among class-13 -amylase sequences (Fig. licheniformis and B.Zn) two-metal center of PWA (Fig. However. kodakaraensis. woesei. respectively. whereas the coordination geometry of the second differs. require high temperatures of up to 100 °C. indicating that competitive metal inhibition could be a general feature of members of this family. we assume that these groups are protonated and thus are kept positively charged by the nearby carboxylate groups of Asp289 and Glu222. Therefore. These data indicate divergent evolutionary paths for the adaptation of -amylases in Archaea (P. Another specific feature of the PWA structure is the presence of a disulfide bond in domain B that is formed by two adjacent cysteines.Zn) Metal Center-containing -Amylase ring in acarbose) is not directly visible in the PWA structures. a molar excess of calcium almost completely inhibits the catalytic activity of the -amylase from Aspergillus niger (40).Zn) two-metal center in close proximity to the active site cleft. Implications for Biotechnological Processes—Amylases. Although in all structures of glycosylhydrolase class-13 amylases known so far. in contrast to the two calcium sites in BLA. However. whereas no metal ion is found in the same site in the PWA Tris structure. the catalytic activity of PWA at high temperatures is dramatically reduced. its affinity for different metal ions may vary. 2). the precise molecular role of this disulfide bridge in PWA substrate catalysis with respect to thermostability still remains to be defined experimentally.Zn)-binding Site Is Essential for PWA Activity. Even in the more closely related Archaea sequences from Pyrococcus kodakaraensis and Thermococcus sp. One belongs to the hyperthermophile B. This sequence motif may also be indicative of the previously identified close relations between Archaea and plant -amylases (47). If the zinc site of this two-metal center is abolished by replacing its cysteine ligand (Cys166). This site superimposes well with the (Ca. woesei has evolved this structural property as a unique evolutionary adaptation most probably to retain full PWA activity in its extremophilic living condition. Not only is PWA superior to other -amylases with respect to thermostability. these -amylases have evolved as either a homo(Ca. which was crystallized in the absence of zinc. however. The PWA (Ca. P. and Thermococcus sp. To date. which is common to many class-13 -amylases. At present. along with other starch-hydrolyzing enzymes like pullulanases and glucoamylases.or a hetero-(Ca. rigidification of the active site area by additional conformational constraints imposed by the presence of such a disulfide bridge may be required for in vivo catalytic activity. whereas it has little effect on the PWA catalytic activity. Topt 90 °C) to high temperature environments. Cys153 and Cys154. on November 14. specifically methanol dehydrogenase from Methylobacterium extorquens (48.. this cysteine is replaced by an alanine. the -amylases from these organisms display full catalytic activity and stability only if calcium is added.Zn) two-metal center. The other known -amylase structure with a two-metal center is the meso-stable -amylase from barley. stearothermophilus are used commercially in the liquefaction process of starch due to their high thermostability (5). despite the conserved nature of the 1 active site pocket in -amylases. Some of these processes. The close proximity of the zinc site of the two-metal center in PWA and the Cys153–Cys154 disulfide bridge suggests a possible joint requirement for -amylase activity under the physiological conditions of P. 4A). licheniformis. connecting residues that are adjacent in sequence. under the specific living conditions of P. licheniformis. in PWA. Such an atypical disulfide bridge. is rare in available protein structures. niger (40). In this work. The three structures in the presence of different inhibitors (zinc.Zn) two-metal center. the calcium in domain B is coordinated by not more than six protein ligands. acarbose/zinc. These data are reflected by the presence of calcium in the 1 active site pocket in the structure of the -amylase from A. One loosely related plant -amylase sequence from 9883 Oryza sativa (24% sequence identity) also contains the same molecular arrangement. mostly the -amylases from B. the PWA structure reveals a novel zinc-binding site in close proximity to the calcium-binding site previously established to be essential for catalytic activity and stability (13). This sequence motif is confined to a limited number of Archaea -amylase sequences. indicating loss of thermostability (13).Zn) two-metal center in PWA. which are used in subsequent starch-processing reactions. woesei) and bacteria (B.Ca). Both two-metal centers share the conserved calcium-binding site (site I in BLA) (27). and pharmaceutical industries (4. available cysteine mutations do not influence thermostability and catalytic activity significantly under the conditions of the present in vitro measurements (13). indicating that this site is lost. We speculate that. Thus. no systematic structural and functional studies are available in which the ability of zinc to act as a competitive inhibitor of -amylases from different organisms was investigated. thus providing a structural rationale for the high binding affinity of the latter enzyme. T. only two other structures of -amylases with a two-metal center are available. In addition. Unfortunately.org by guest. woesei at high temperatures. but it also lacks an exogenous calcium requirement for full catalytic activity (Table III) (10. 46). Comparison of -amylase sequences (data not shown) and use of Cys166 as an indicator denote that the zinc site of the two-metal center is present only in P. but Is Not Conserved—We have solved the first structure of an -amylase that comprises a mixed (Ca. thus stimulating investigations into alternative -amylases that do not require addition of metal ions during enzymatic processes at the industrial scale. utilizing zinc as a positive and negative regulator in a concentration-dependent manner. However. 6) and compose 30% of the current industrial enzyme production. which binds two calcium ions that are probably bridged by a sodium ion (27). have widespread applications in the food. Activation of PWA by traces of zinc is. hydrothermalis. To date. The PWA structures offer a strong base upon which to further engineer properties of Downloaded from www. and Tris) provide a molecular rationale for previous biochemical analyses of PWA (13) and our current data (Table III) indicating specific and tight binding of zinc and calcium. 13). 6). 2010 . it is coordinated by seven protein ligands. woesei and its close homolog Thermococcus hydrothermalis (84% sequence identity). the second calcium site of the barley -amylase structure does not superimpose with the second site of the (Ca. superseded by the competitive active site inhibitory effects of this metal. thereby conferring comparable specific PWA binding.Structure of a (Ca.

. E. A. Tamada. Greenwood.. 319 –326 Butz. Downloaded from www. M. and Moffat.. Sidhu. Davies. T. Svendsen. 108 –118 38.. 280.. C. (2002) Curr. M. H.. L. 271–313 43. A.. Saint.. Aghajari. (1976) Appl. T. Turkenburg.. Chem. H. Cascio.. U.. and Fischer. Svensson. 19.. Jensen.. Sect. (2002) Biochemistry 41.. A. 905–921 Jones. F. G. A. and Haye. J.. S. C. G.. K. R. and Anthony.. S. G. J. W. F. 3.. Wilson. and Haser.. P. T. S. (2002) J. Bourne. Kuszewski. (1986) J. (2001) Biotechnol. 25273–25278 37. G. (1998) Protein Sci. Crystallogr. B... T. M. T. Minematsu.. 253–259 Savchenko. Astier. (1993) J. Flagmeyer. L. M. K. Dauter. Biol.. Y. Henriksen. 10... S.. 12... M. (2001) Mol.. E. Derewenda. Swift. Vieille. C. 272. (1991) Acta Crystallogr.. Huber. Janecek. 67–97 Pujadas. 564 –572 35. 853– 859 Janecek. Dodson. 4273– 4280 34. S. and Kuroki.. Sierks. Chem. 6193– 6201 Niehaus. Day. and Zeikus. J. Solovicova. B.. and Lamzin. L.. Honda. 151–160 Henrissat. 102–105 49.. Z. Adv. Chromatogr. S. and Svensson. 51. T...... R.. 17. and Gajhede.. Wilson. (1994) Nat. E. 234. 309 –316 Feese. and Haser.. Juy.. M. Kahler. Hostinova. J. H. J. B. Mol. Biol. C. Mi¨ crobiol. D. Biophys. Sect. D Biol. 22.org by guest. S. D. Mol. E. C.. G. C. 281–292 28. M. on November 14. Simonson. C. D. (1998) Acta Crystallogr. J. W. Wilson. G. Wang. and McPherson. Lehmann... Mol. Biol. 19.. A. (2001) Acta Crystallogr. G. 421– 426 48. MacGregor... 15.. P. C. P. Cambillau.. 649 – 659 47. Microbiol. 23. 760 –763 De la Fortelle.... J. Legge. Roth. and Haser. Navaza. Hondo. Environ.. Biol. K. Sect. Wang. (1994) Acta Crystallogr.. Monsan. L.9884 Structure of a (Ca. Harlos. R. Blake... Bernfeld. A. and Kjeldgaard. A. V. and Woldike. (2001) BioMetals 14.. Crystallogr. 419. 157–163 26. Bisgaard-Frantzen. and Sander. 451– 464 Vieille. R. Methods B 161.. Reinert.. J. 301. D Biol. G. B. Vallee. 14. 7. M. K. H. 791– 805 29. A.. Auld.. Rice. Biol. C. DeLano. Heitmann. P. G. S. C. Boel. S.. Sevcik.. 276. Biol... M. Kobayashi. 4. and Antranikian. Vieille. GrosseKunstleve. and Warren.. (1999) Biochemistry 38. D Biol. J. S. Dauter. Remaud-Simeon.. and Dijkhuizen. Declerck. (1996) Eur.. 189. (2000) J. 1. 16335–16342 Dong. and Antranikian. Chromatogr. 51– 60 39. 182.. 104 –121 46... Keitel. Segawa. and Wiegand. Rodenburg. (1959) J. Harding. Biol. Z. T. D. Henrissat. J.. M. A. 123–138 27. M. Evol.. A. (1997) Methods Enzymol..Zn) Metal Center-containing -Amylase Crystallogr. 21. T.. (1997) Prog. Leverkusen. Goel. REFERENCES 1. Bertoldo. C. Ohmura. Abe... R. Komeda.. Stein. Beier. H. K. Auld for helpful discussions on the interpretation of metal-binding sites.. 57. M.. A. In particular. Miura. (1997) Methods Enzymol. C. and Antranikian. and Palau. Biochem.. 261–278 Van der Maarel. Kang. G. J. Reibetanz. 63. G. J. S. G. Vorgias. 238. W.. Spemann. J. and Withers. and Antranikian.. (1995) Structure 3. B. Svensson. 323–327 Otwinowski. W. Mol. J.. A. C.. Biol. B. T. G. M. 737. M. G. S. P. (2000) Biochemistry 39. 67. 276.. Ghosh. D Biol. G.. M. R. Chem.. W.. 961–974 51.. 9099 –9107 33. 307–326 Collaborative Computational Project Number 4 (1994) Acta Crystallogr. G. C. Biol.. R. and von Hippel. E. Sumerwell. D. N. 235. Instrum. Wilson. Struct. M.. Kadziola.. T.. J. B. A. 1– 43 Jorgensen. M. R. Machius.. Stezowski. V. 6. and Haser. Harlos.. K. (2001) J. S.. 20. P.. A. Diehl. Ghosh. O. Nilges.. T. Troger. Rev.. J. Savchenko. (2002) Biochemistry 41. 6244 – 6249 41.. D. R.. L. A. E. Christensen.. B. Biotechnol. Protein Chem. W.. Biochem. F.... (2000) Biochemistry 39. 2010 18. J. 233. G. K. R. Knaute. which are well suited to optimize the properties of PWA for biotechnological applications. and Blake... 472– 494 Brunger. (2000) Nucl. R. 2123–2126 40. S. 53.. Biol. and Hartman. Skov. 54. M. and Davies. Jespersen. Clore. L. Feller.. 13. Kadziola. M. M. and Bricogne. J. Sarcabal.. M. S. (1998) Acta Crystallogr. 8. M. 5. G. Szymanski. 16.. M. A 47. F. J... Opin.. L.. De Montalk.. 3569 –3576 Linden. D. C. F.. and Kakehi. Crystallogr. 177–187 50. Sect. 54. 11. Cowan. Hirose. J. P.. 31. Sect. Biol. N. F. E. G. 8385– 8392 31. (1999) Appl. Irshad. (1981) Phytochemistry 20. T. B. Gilles. Jiang. Sixma. (1998) Structure 6. C. and Zeikus.. Gasperik. (1987) J. and Gorisch. D. A.. (1997) J. E. Pannu. J.jbc. Kato. J.. Mol... A. A. Dauter.. Biol. E. Biol. J. 401– 411 44.. N.. (1997) Appl. A. (2000) J. 6. C. Aghajari. T. Uitdehaag. Mol. Microbiol. W. B. A.. F. Leemhuis. L. (1995) Structure 3. 1560 –1584 45. Chem. P.. N. Lawson.. Vallee. Vogt.. and Davies.. 110 –119 Brunger. Zou. G.. J. H.. J. S.. Mirza. (1994) J. .. J. (2000) J.. K.. Goodwin. Y. Moller (Bayer AG. G. H. Jamieson. B. J.. K. Mol. 2901–2905 42. S. Overall. Adams. Brzozowski. Acknowledgments—We thank E. and Dauter.. 94. H. M. G. 38 –54 Eichler. J.. P. Y. Anthony. Borchert. D. Gros. Biol. Mol.. G. Crystallogr.. Thim.. H. Petersen... Gerday. Kato. G. N. Braun. 24. Biotechnol. Brzozowski. P. S. V. 65. Sect. and Zhu. Hohne. 12. 48. D Biol. Environ. (1990) Biochemistry 29. L. Niehaus. (1998) Structure 6. Z. K. Rydberg. A.. G. they reveal two metal-binding sites (zinc and calcium) with different functions.. and Zeikus. C. Read. 7. V. van der Veen.. 113–130 Gill. 18. 448 – 455 25. J. L.-Y. (1993) J. M. 239. Brady. and Henrissat. 297. (1994) J. 711–729 Studier. T. H. B.. Crystallogr.. Holm. and Payan. 4778 – 4791 32. K. Avezoux. ¨ Germany) for kindly providing acarbose and D.. 137–155 Bertoldo. Z. (1955) Methods Enzymol. A. 472– 475 ¨ Perrakis. Brayer. S. Y. Y. K. (1997) Acta 9.. 50. Larson. N. and Minor. Nguyen.. Belarbi. Maurus. (1999) J. G. Evol. 561–569 30.. A. M... Z. C. B.. M. Marchis-Mouren. W. (1991) Biochem.. A 50.. 2. 854 – 866 36. Brzozowski. Leveque.. Willemot. M... Borchert. R. M. Sect. 276. M. and Sharma. 149 –158 PWA that are more conducive to potential applications in industrial processes. R. (2001) Microbiol. (1989) Anal. C. Mol. T. 1. T.. (1992) Nature 355. S.