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

Anni LindenDagger , Olga MayansDagger §, Wolfram Meyer-KlauckeDagger , Garabed Antranikian, and Matthias WilmannsDagger ||

From the Dagger  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

Received for publication, November 6, 2002, and in revised form, December 11, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The crystal structure of the alpha -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 alpha -amylase bound 1 and 4 molar eq of zinc and calcium, respectively. The structure reveals a novel, activating, two-metal (Ca,Zn)-binding site and a second inhibitory zinc-binding 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 alpha -amylase, in contrast to commercially available alpha -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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha -Amylases (alpha -1,4-D-glucan 4-glucanohydrolase, EC 3.2.1.1) are endo-acting hydrolases that randomly cleave the alpha -1,4-glycosidic 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 alpha -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 alpha -amylases from organisms inhabiting environments that span a large temperature range, from psychrophilic to hyperthermophilic (3). These alpha -amylases share a common three-domain fold with the catalytic activity located on the C-terminal face of a central (beta alpha )8-barrel, domain A. The 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 alpha -amylase activity (8), no other structures of archael class-13 alpha -amylases are known to date. The alpha -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 alpha -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 alpha -amylases, PWA activity does not require the addition of metal ions. Due to its superior properties, exceeding those of alpha -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 alpha -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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -amylase gene from P. furiosus (10) and to the P. woesei amylase sequence released to the NCBI Protein Database by Lu et al.2

Purification and Concentration Determination-- The recombinant alpha -amylase gene product was purified from catalytically active inclusion bodies (12). Inclusion bodies were solubilized in 0.12 M Britton and Robinson buffer (pH 12) and purified by hydrophobic interaction chromatography using a phenyl-Superose HR 5/5 column (Amersham Biosciences, Freiburg, Germany). The protein was eluted in 0.02 M triethanolamine containing 30% (v/v) isopropyl alcohol (pH 7.2). Fractions containing amylolytic activity at 99 °C were pooled and further purified by size-exclusion chromatography using a Superdex 200 16/60 prep-grade column. The amylase was eluted in 0.02 M triethanolamine (pH 7.2) and concentrated to a final concentration of 5.5 mg/ml in an Amicon ultrafiltration device (Millipore Corp., Eschborn, Germany). The concentration of the enzyme preparation was determined by estimating the extinction coefficient according to the method of Gill and von Hippel (16).

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). Using this technique, all elements besides those lighter than sodium can be detected in a single scan at a minimum weight limit of ~1-10 ppm. The measurements were calibrated using the nine sulfur atoms of the PWA sequence (five cysteines and four methionines) as an internal standard. 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. The samples (~2 µl, 1 mg/ml) were dropped onto sample holders covered with a 0.9 µm polyethylene terephthalate foil. A sample area of ~1 mm2 was scanned with the proton microbeam, and the characteristic x-rays were detected using a germanium detector. A total charge of ~0.17 coulomb was applied within 20 min, ensuring that no elemental loss (sulfur or metals) due to thermal stress occurred, which would otherwise have changed the x-ray signal intensity. The number of metals per molecule was calculated from their calibrated signals with reference to the calibrated sulfur signal. 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.

Crystallization-- Purified recombinant PWA was crystallized at a concentration of 5.5 mg/ml by the hanging drop vapor diffusion method. PWA·Zn was grown from 1 µl of protein solution and 2 µl of 0.1 M MES (pH 6.5) containing 0.01 M Zn2(SO4)·7H2O and 25% (v/v) polyethylene glycol monomethyl ether 550 at 19 °C. The PWA·Ac/Zn complex was grown from 1 µl of acarbose solution (11 mg/ml), 1 µl of protein solution, and 1 µl of 0.1 M sodium cacodylate (pH 6.5) containing 0.05 M Zn(OAc)2 and 35% (v/v) 2-methyl-2,4-pentanediol at 25 °C. The PWA·Tris complex was crystallized from 1 µl of acarbose solution (11 mg/ml), 1 µl of protein solution, and 1 µl of 0.1 M Tris (pH 8.5) containing 0.05 M MgCl2 and 40% (v/v) ethanol. 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 1.0 mM CH3HgCl for 1.5 h at 19 °C prior to x-ray data collection.

X-ray Data Collection, Processing, 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. For PWA·Tris, 40% (v/v) ethanol and 31.5% (v/v) polyethylene glycol 400 were used as a cryoprotectant. For PWA·Ac/Zn, no transfer into a cryoprotectant was carried out. All crystals were mounted onto nylon cryo-loops (Hampton Research, Riverside, CA) and shock-frozen in a nitrogen stream at 100 K. X-ray data sets for the PWA·Zn, PWA·Ac/Zn, and PWA·Tris complexes were recorded up to 2.2-, 2.0-, and 1.5-Å resolution, respectively. An anomalous diffraction data set to 2.5-Å resolution was collected from a PWA mercury derivative isoform at a wavelength above the L-III adsorption edge of mercury. Further data statistics are presented in Table I. The data were processed, merged, and scaled using the HKL program suite (18).

Structure Determination, Refinement, and Model Evaluation-- Programs used for the subsequent calculations were from the CCP4 program suite (19), unless stated otherwise. Initial phases for PWA·Zn were obtained using single isomorphous replacement including the one-wavelength anomalous scattering contribution (SIRAS). Heavy atom positions were determined from mercury derivative difference Patterson maps using the RSPS program. From three major binding sites, initial phases were calculated and refined using SHARP (20) up to 2.5-Å resolution. Phases were improved by solvent flattening with SHARP. An initial map calculated in CNS (21) was used to build the model of the molecule using the interactive graphics program O (22). The model was traced with the help of Bacillus licheniformis alpha -amylase (BLA) as a template. The final model was built in a consecutive cycle of crystallographic refinement using CNS and manual rebuilding. A total number of 25,967 reflections in the resolution range of 20-2.22 Å were included in the refinement, with 3.8% (995 reflections) set aside for cross-validation (23). The initial R-value was 44.2%, and the free R-factor was 42.1%. After several cycles of positional and restrained individual B-factor refinement, 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 sigma . 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. Rotation and translation functions were calculated with AMoRe (25) using x-ray data from 10 to 2.5 Å, resulting in a single solution with correlation coefficients of 68.6 and 80.1% and R-factors of 39.4 and 33.5% for PWA·Tris and PWA·Ac/Zn, respectively. A test set of 1686 reflections (4.7%, PWA·Tris) and 1196 reflections (1.7%, PWA·Ac/Zn) for the calculation of Rfree was excluded during refinement in CNS. Initial phases of the PWA·Tris model were applied to automated model building using ArpWArp for model completion. The models were verified and corrected, and acarbose and Tris molecules were built manually using the graphics software program O. Solvent building and subsequent refinement were performed as described above.

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. 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. 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. 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 12.4 keV (lambda  = 0.91 Å), which is above the k-adsorption edge of zinc of 9.59 keV (lambda  = 1.28 Å).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Overall Structure and Ligand Composition-- We have solved the structure of PWA in three different forms using experimental phases from a mercury derivative. The structures are in the presence of three different inhibitors: 1) zinc (PWA·Zn); 2) the specific substrate analog acarbose and zinc (PWA·Ac/Zn); and 3) Tris, which was used as a buffer for crystallization trials in the absence of zinc (PWA·Tris). They have been refined to 2.2-, 2.0-, and 1.6-Å resolution, respectively. PWA displays the canonical glycosylhydrolase class-13 fold that is composed of three domains, A-C (Fig. 1). Comparison of the overall structure of PWA with that of other known alpha -amylases using the program DALI (26) revealed the highest structural similarity to the homologous enzymes from the hyperthermophile B. licheniformis (BLA; 29% sequence identity; root mean square deviation of 2.5 Å based on comparison of the Calpha backbone trace) and from barley (28% sequence identity; root mean square deviation of 1.8 Å) (Fig. 2).


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Fig. 1.   Overall crystal structure of PWA complexed with different ligands. The cylinders and arrows represent helices and beta -strands, respectively. Domains A-C are colored cyan, magenta, and brown, respectively. For clarity, the beta -strands composing the beta -barrel of domain A are colored gray. Bound metals (zinc, green; and magnesium and calcium, orange) and acarbose molecules are indicated and numbered according to Table II. The backbone of the acarbose molecules is yellow; oxygen atoms are red; and nitrogen atoms are blue. A, top view of the PWA·Ac/Zn complex. The active site cleft at the front face of the PWA molecule contains two inhibitors with partial occupancies, the first of which is acarbose and the second of which is a coordinated zinc ion, which is virtually identical to the nitrogen position of the 4-amino-4,6-dideoxy-alpha -D-glucose ring of acarbose. The two inhibitors are superimposed onto each other. B, PWA·Ac/Zn rotated 90° along a horizontal axis with respect to the orientation in A. C, PWA·Tris shown from the same orientation as in A. Three zinc-binding sites (Zn3, Zn5, and Zn6) of PWA·Ac/Zn are replaced by magnesium ions in PWA·Tris (Mg3, Mg5, and Mg6). In PWA·Tris, no metal is found in site 4 of the PWA·Ac/Zn structure.


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Fig. 2.   Structure-based alignment of PWA sequences. The secondary structure assignments for PWA are shown as colored cylinders (310-helices and alpha -helices) and arrows (beta -strands) above the aligned sequences. AVA, Hordeum vulgare alpha -amylase (AMY2) chain A (Protein Data Bank code 1AVA) (46). The Protein Data Bank code for BLA is 1BLI (27). The positions of amino acid residues coordinating the zinc ion in domain B (site 1; cf. Table II) are highlighted in red; those coordinating calcium (site 2; cf. Table II) in domain B are colored yellow; and the cysteines forming disulfide bonds are colored green. The seven invariant amino acid residues are marked in black. The alignment was carried out with DALI using the main chain positions of each coordinate set.

As in other alpha -amylases, the central domain A, covering residues 1-109 and 170-340, is folded as a (beta alpha )8-barrel and contains the active site at its C-terminal face. Domain B (residues 111-169) inserts between beta -strand 3 and alpha -helix 3a of domain A, thus forming part of the active site cleft. Its secondary structure is limited to two short beta -strands, forming a small antiparallel beta -sheet and a short 310-helix (Figs. 1 and 2). At their interface, domains A and B comprise a novel (Ca,Zn) metal center, which is described further below (see Fig. 4). 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. Covering a range of 58 residues only, domain B of PWA is one of the smallest alpha -amylase B domains, whereas other members of the family span >100 residues (2, 3). Among those with a known structure, domain B of BLA is most similar (root mean square deviation of 2.3 Å based on the Calpha backbone) (27) to the corresponding domain of PWA. In contrast, domain B of BLA contains 43 additional residues that form a second beta -sheet (27). The C-terminal domain C (residues 341-435) is arranged in an eight-stranded antiparallel beta -sheet containing a Greek key motif. The function of this domain still remains unclear.

We initially identified the nature of the bound metal ions by PIXE. For these experiments, PWA was expressed heterologously in E. coli and purified in the absence of exogenous metals except for sodium. The PIXE data revealed the presence of 1.1 ± 0.4 molar eq of zinc and 4 ± 2 molar eq of calcium bound to PWA. In the PWA crystal structures, 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). Based on these data, the PWA structures show excessive metal binding; all three structures have five metal-binding sites in common (Table II). The two crystal forms grown in the presence of zinc (PWA·Zn and PWA·Ac/Zn) reveal two additional metal sites, bringing the total in these structures to seven. 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). In the PWA·Tris structure, solved from crystals grown in the absence of zinc, but in the presence of magnesium, only one site showed a significant anomalous signal and therefore was refined as a zinc site (Table II). The remaining four sites, without an anomalous signal, but retaining strong positive difference electron density, when refined as an ordered solvent (>10 sigma ), were interpreted to be calcium or magnesium sites. Thus, the stoichiometry of calcium (or magnesium) and zinc sites observed in the PWA·Tris structure is in good agreement with the PIXE data. In contrast, in the PWA·Zn and PWA·Ac/Zn structures, all sites except one showed a significant anomalous signal and were therefore refined as zinc sites. The remaining site was interpreted as a calcium site. These metal sites will be referred to as activating sites (Table II, sites 1 and 2), an inhibiting site (site 7), and other sites (sites 3-6).

                              
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Table I
X-ray data and structure refinement statistics

                              
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Table II
Metal-binding sites in PWA of different crystal forms
The values for the sigma  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." The sigma  values for the anomalous peaks (ano) were obtained from the anomalous difference electron density maps calculated using fast Fourier transformation.

Chemically Unrelated Inhibitors Competitively Bind to the Active Site-- We quantified the inhibitory effects of a number of established alpha -amylase active site ligands, including the transition state analog acarbose and the buffer Tris (Table III). To link the observed tight binding of calcium and zinc to a potential role in activity regulation, we also assessed the regulatory properties of a number of metal ions (Table III). Except for copper (full inhibition in the presence of 3 mM Cu2+), zinc had the strongest inhibitory effect. In the presence of >= 3 mM zinc, residual PWA activity was <10%. In contrast to many other characterized alpha -amylases, including commercially available BLA (10, 13), PWA was not activated significantly by an excess of calcium (Table III). Based on the measured inhibition data, we selected three ligands (acarbose, Tris, and zinc) for crystallographic characterization of the PWA active site (Fig. 3).

                              
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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 %). The measurements were performed according to Bernfeld (51) in 0.05 M sodium acetate and 1% (w/v) starch (pH 5.5). Each reaction was carried out with 11.5 ng of PWA for 4 min at 94 °C.


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Fig. 3.   Active site of PWA with bound acarbose and zinc (PWA·Ac/Zn; A and B), Tris (PWA·Tris; C and D), and zinc (PWA·Zn; E and F). A, C, and E, structures of active site residues in the presence of bound ligands. Each Fo - Fc difference electron density map (green) in the absence of ligands (A, acarbose; C, Tris; and E, zinc) is contoured at 2.0, 2.0, and 4.5 sigma , respectively. In E, the anomalous difference peak (red) is contoured at 3.7 sigma . B, D, and F, schematic representations of the ligands bound to the active site. Hydrogen bonds are shown by dashed lines. Zinc ions and solvent molecules are shown as green and gray spheres, respectively. Solvent molecules mediating protein-inhibitor interactions are indicated in B, D, and F; for clarity, Tyr62, Phe159, and Tyr199 are not shown.

Acarbose is a pseudotetrasaccharide inhibitor consisting of a valienamine unit at the nonreducing end linked to 4-amino- 4,6-dideoxy-alpha -D-glucose, which is fused to maltose. In the PWA·Ac/Zn structure, resulting from co-crystallization of PWA in the presence of acarbose and zinc, one acarbose molecule is bound to the active site at the C-terminal face of the (beta alpha )8-barrel of domain A. It interacts with a number of highly conserved residues that reside in loops connecting beta -strands 4, 5, and 7 of the (beta alpha )8-barrel with the subsequent helices (Figs. 2 and 3, A and B) (2, 3, 28). Three of its four sugar rings (A-C), comprising the acarviosine residue and a linked glucose ring, are visible; but the forth unit accounting for the last glucose unit at the reducing end of the molecule is not. The location and orientation of the acarbose inhibitor within the active site of PWA resemble previous data from several alpha -amylase·acarbose complexes. However, in contrast to previous observations (29-33), PWA does not display acarbose transglycosylation activity, indicating that PWA is not catalytically active under crystallization conditions. We reasoned that the low temperature used for crystal growth of this hyperthermophilic alpha -amylase has been sufficient to inhibit any transglycolytic catalysis within the crystal.

We noticed that residual anomalous difference electron density remained at the nitrogen position of the 4-amino-4,6-dideoxy-alpha -D-glucose ring within the active site of the refined PWA·Ac/Zn complex (Fig. 4C). Therefore, a zinc ion was placed into this position as an alternative inhibitor (Table II, site 7). Zinc, like the amino group of the 4-amino-4,6-dideoxy-alpha -D-glucose ring of acarbose, is bound by the carboxylate group of Glu222. The occupancies of the two inhibitors, acarbose and zinc, were refined to final values of 0.6 and 0.4, respectively. 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. 3, A and B; and 4C). On the other hand, if acarbose was added in 100 mM Tris buffer without zinc, a Tris molecule entirely replaced acarbose in the active site of PWA (PWA·Tris). Tris is bound by residues that interact with the valienamine (Arg196, His288, and Asp289) and 4-amino-4,6-dideoxy-alpha -D-glucose (Glu222 and Asp289) groups of acarbose in the PWA·Ac/Zn (Fig. 3, C and D), confirming previous structural data from a number of alpha -amylase·Tris complexes (32, 34-36) and demonstrating its function as a potent competitive inhibitor (34, 37, 38).


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Fig. 4.   Unique metal-binding sites in PWA. A, structure of the two-metal (Ca,Zn)-binding site in PWA (cf. Table II, sites 1 and 2). The zinc ion (gray sphere) is coordinated by two histidines (His147 and His152), one cysteine (Cys166), and an ordered solvent molecule (red). The calcium ion is coordinated by the conserved residues Asn110, Asp155, Gly157, Asp164, and Gly202 (cf. Fig. 2) and a solvent molecule. The Fo - Fc difference and anomalous difference maps are colored as described in the legend to Fig. 3 and contoured at 4.5 and 33 sigma , respectively. B, structure of the variable PWA metal-binding site in domain C (cf. Table II, site 3). Left panel, zinc (green sphere) is coordinated by three negatively charged amino acid side chains (Asp347, Asp349, and Glu350) and a solvent molecule (red sphere). Right panel, in the PWA·Tris structure, a magnesium ion is coordinated by the same side chain residues as zinc in the left panel. However, two additional solvent molecules lead to a different overall coordination geometry. C, shown is the structure of the partially occupied inhibitory zinc-binding site in the -1 active site pocket of PWA, which is superimposed on the bound acarbose ligand. The residual anomalous difference electron density at the zinc position is shown at a contour level of 3.2 sigma .

In the third crystal form (PWA·Zn), zinc binds to the carboxylate group of the same residue (Glu222) (Fig. 3, E and F; and Table II, site 7) that binds to the 4-amino-4,6-dideoxy-alpha -D-glucose group of acarbose (Fig. 3, A and B) and Tris (C and D). The carboxylate group of Asp289 interacts with the zinc ion by a solvent-mediated hydrogen bond. 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. However, the PWA·Zn structure permits a more detailed description of the coordination geometry of the zinc ion. 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. 3, A and B). Two additional solvent molecules are located at O-4 and O-6 of the acarbose complex and one near O-3, thus substituting the oxygen atoms of the glucose ring of the natural substrate bound to the -1 subsite. Soaking of the PWA·Zn crystal form in a solution containing 15 mM acarbose at pH 6.5 confirmed partial replacement of zinc by the inhibitor (data not shown). Thus, the PWA·Zn structure provides, for the first time, a molecular rationale for previous observations (10) and our data (Table III) showing how zinc, at concentrations >5 mM, entirely inhibits the amylolytic activity of PWA. We anticipate that the strong inhibition of many alpha -amylases by zinc (39) is via the same binding to the -1 site as observed in PWA·Zn. In contrast, the PWA structures do not indicate that magnesium binds to the active site, confirming previous biochemical observations that magnesium does not inhibit enzyme activity (10, 11).

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, irrespective of specific crystallization conditions and ligand binding to the active site. Therefore, it is most likely that the observed metal ions originate from the medium that was used for heterologous expression of PWA in E. coli. The two metal sites are separated by 7.3-7.4 Å (Fig. 4A and Table II, sites 1 and 2) and are located within the interface of domains A and B. Only one of the two sites showed an anomalous signal under the energy conditions used for x-ray data collection (Table I), thus indicating a hetero-population of metals at this site.

Generally, class-13 glucosidases contain a common calcium-binding site (40), which is essential for their catalytic activity (2, 41). In the PWA structure, this site is matched by a peak without an anomalous signal, confirming it as common calcium site. The calcium ion is coordinated by seven protein ligands in a distorted octahedral geometry involving the conserved residue Asn110 from loop beta 3 (domain A)-beta 1 (domain B), three residues from the loop preceding alpha -helix 3a of domain A (Asp155 as bidendate ligand, Gly157, and Asp164), Gly202 from a 310-helix between beta -strand 4 and alpha -helix 4 of domain A, and one ordered solvent molecule (Fig. 4A). Thus, the number of protein ligands in this calcium-binding site exceeds those found for the same site in other class-13 alpha -amylases (2).

In contrast to the first metal site (Table II, site 1), there is a strong anomalous contribution at the second site (site 2). 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, confirming previous data (13). The zinc ion is coordinated by ligands in a distorted tetrahedral geometry, including the imidazole groups of His147 and His152, the sulfhydryl group of Cys166, and an ordered solvent molecule. This coordination geometry is typical for protein·zinc complexes (42, 43). If the cysteine ligand (Cys166) is replaced, thereby abolishing the zinc site of this two-metal center, the catalytic activity of PWA at high temperatures is dramatically reduced, indicating loss of thermostability (13). Thus, both sites of this two-metal center have a common role, to stabilize a catalytically active conformation in PWA at high temperatures.

Additional Ligand-binding Sites-- The high resolution data of the three PWA structures have allowed the identification of additional ligand-binding sites (Table II). However, at present, 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. Therefore, only a brief account of these sites is given.

Apart from the acarbose site within the PWA active site that is observed only in the PWA·Ac/Zn structure, 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. 1). The second acarbose-binding site, with three acarbose rings visible (Ac-II), is located in a slight depression formed by the 310-helix preceding beta -strand 1 of domain A, the loop connecting alpha -helix 6b and beta -strand 7 of domain A, and the loop connecting alpha -helix 8b of domain A and the first beta -strand of domain C (Fig. 1). A similar carbohydrate-binding site was reported for a chimeric Bacillus amyloliquefaciens/licheniformis alpha -amylase (32). 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. 1). This site corresponds to the so-called accessory carbohydrate site reported for the pig pancreas alpha -amylase structure (44). The fourth acarbose-binding site (Ac-IV) is located at the surface of the domain A/C interface (Fig. 1), again with three sugar rings visible. These three rings interact with residues from alpha -helix 8b of domain A and beta -strand 5, the following loop of domain C, and the C terminus of the protein molecule.

The presence of an anomalous difference at the remaining metal-binding sites (Table II, sites 3-6) depends on the presence of zinc during crystallization, suggesting that metal binding at these sites is weaker and less selective than at the (Ca,Zn) metal center, where the presence/absence of the anomalous difference does not depend on crystallization conditions. All three PWA structures display an additional metal-binding site (Table II, site 3) at the loop connecting beta -strands 1 and 2 of domain C (Fig. 4B, left panel). In the PWA·Ac/Zn and PWA·Zn structures, a zinc ion was modeled in this site, which is coordinated by the three carboxylate groups of Asp347, Asp349, and Glu350 in a trigonal planar geometry. In the PWA·Ac/Zn structure, this site is also liganded by an ordered solvent molecule. In the PWA·Tris structure, in contrast, 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. 4B, right panel). The structure suggests that this metal-binding site generally serves a stabilizing role, which may be further enhanced by a nearby disulfide bridge connecting Cys388 and Cys432. Two other metal-binding sites within the interfaces of symmetry-related molecules have been found in the PWA structures (Fig. 1 and Table II, sites 5 and 6). One additional metal-binding site in domain A (Table II, site 4) is found only in the presence of zinc, involving at least one lysine residue as ligand, which is a rare feature in known protein crystal structures (43). Overall, the total number of metals coordinated by a single PWA alpha -amylase molecule exceeds the number of bound metals previously reported for any other class-13 glycosylhydrolase. 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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Zinc Is a Competitive Active Site Inhibitor That Binds to the Conserved -1 Active Site Pocket-- Acarbose and related sugar units containing alpha -amylase inhibitors bind to at least three specific active site pockets in class-13 alpha -amylases. 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. In PWA, this site not only binds organic compounds like acarbose and Tris, but also serves as an inhibitory metal-binding site of limited specificity. The two chemically related metals Cu2+ and Zn2+ show comparable inhibition of PWA catalysis, 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-4,6-dideoxy-alpha -D-glucose ring in acarbose) is not directly visible in the PWA structures, we assume that these groups are protonated and thus are kept positively charged by the nearby carboxylate groups of Asp289 and Glu222, thereby conferring comparable specific PWA binding.

The -1 pocket residue ligands are highly conserved among class-13 alpha -amylase sequences (Fig. 2), indicating that competitive metal inhibition could be a general feature of members of this family. To date, no systematic structural and functional studies are available in which the ability of zinc to act as a competitive inhibitor of alpha -amylases from different organisms was investigated. Interestingly, a molar excess of calcium almost completely inhibits the catalytic activity of the alpha -amylase from Aspergillus niger (40), whereas it has little effect on the PWA catalytic activity. These data are reflected by the presence of calcium in the -1 active site pocket in the structure of the alpha -amylase from A. niger (40), whereas no metal ion is found in the same site in the PWA·Tris structure, which was crystallized in the absence of zinc. Thus, despite the conserved nature of the -1 active site pocket in alpha -amylases, its affinity for different metal ions may vary.

The PWA (Ca,Zn)-binding Site Is Essential for PWA Activity, but Is Not Conserved-- We have solved the first structure of an alpha -amylase that comprises a mixed (Ca,Zn) two-metal center in close proximity to the active site cleft. The three structures in the presence of different inhibitors (zinc, acarbose/zinc, 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. If the zinc site of this two-metal center is abolished by replacing its cysteine ligand (Cys166), the catalytic activity of PWA at high temperatures is dramatically reduced, indicating loss of thermostability (13). Comparison of alpha -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. woesei and its close homolog Thermococcus hydrothermalis (84% sequence identity). Even in the more closely related Archaea sequences from Pyrococcus kodakaraensis and Thermococcus sp., this cysteine is replaced by an alanine, indicating that this site is lost.

To date, only two other structures of alpha -amylases with a two-metal center are available. One belongs to the hyperthermophile B. licheniformis, which binds two calcium ions that are probably bridged by a sodium ion (27). This site superimposes well with the (Ca,Zn) two-metal center of PWA (Fig. 4A). Both two-metal centers share the conserved calcium-binding site (site I in BLA) (27), which is common to many class-13 alpha -amylases, whereas the coordination geometry of the second differs. These data indicate divergent evolutionary paths for the adaptation of alpha -amylases in Archaea (P. woesei) and bacteria (B. licheniformis; Topt = 90 °C) to high temperature environments. As such, these alpha -amylases have evolved as either a homo-(Ca,Ca)- or a hetero-(Ca,Zn) two-metal center, respectively. The other known alpha -amylase structure with a two-metal center is the meso-stable alpha -amylase from barley, which shares the highly conserved calcium-binding site and displays an additional calcium-binding site at a distance of ~7 Å (45, 46). However, in contrast to the two calcium sites in BLA, the second calcium site of the barley alpha -amylase structure does not superimpose with the second site of the (Ca,Zn) two-metal center in PWA.

Another specific feature of the PWA structure is the presence of a disulfide bond in domain B that is formed by two adjacent cysteines, Cys153 and Cys154. This sequence motif is confined to a limited number of Archaea alpha -amylase sequences, including those of P. kodakaraensis, T. hydrothermalis, and Thermococcus sp. One loosely related plant alpha -amylase sequence from Oryza sativa (24% sequence identity) also contains the same molecular arrangement. This sequence motif may also be indicative of the previously identified close relations between Archaea and plant alpha -amylases (47). 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 alpha -amylase activity under the physiological conditions of P. woesei. However, available cysteine mutations do not influence thermostability and catalytic activity significantly under the conditions of the present in vitro measurements (13). Such an atypical disulfide bridge, connecting residues that are adjacent in sequence, is rare in available protein structures. Two of these proteins are members of the alcohol dehydrogenase family, specifically methanol dehydrogenase from Methylobacterium extorquens (48, 49) and ethanol dehydrogenase from Pseudomonas aeruginosa (50). The atypical disulfide bridge may stabilize the non-planar semiquinone form of the enzyme's prosthetic group pyrroloquinoline quinone (49). We speculate that, under the specific living conditions of P. woesei at high temperatures, 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. However, the precise molecular role of this disulfide bridge in PWA substrate catalysis with respect to thermostability still remains to be defined experimentally.

Implications for Biotechnological Processes-- Amylases, along with other starch-hydrolyzing enzymes like pullulanases and glucoamylases, have widespread applications in the food, chemical, and pharmaceutical industries (4, 6) and compose ~30% of the current industrial enzyme production. Some of these processes, such as the liquefaction of starch, require high temperatures of up to 100 °C. At present, mostly the alpha -amylases from B. licheniformis and B. stearothermophilus are used commercially in the liquefaction process of starch due to their high thermostability (5). However, the alpha -amylases from these organisms display full catalytic activity and stability only if calcium is added. Unfortunately, the addition of calcium inhibits glucoamylases and destabilizes glucose isomerases, which are used in subsequent starch-processing reactions, thus stimulating investigations into alternative alpha -amylases that do not require addition of metal ions during enzymatic processes at the industrial scale.

Not only is PWA superior to other alpha -amylases with respect to thermostability, but it also lacks an exogenous calcium requirement for full catalytic activity (Table III) (10, 13). Therefore, PWA has been proposed as an alternative to BLA to further improve the efficiency of industrial processes in starch liquefaction (5, 6). In this work, 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,Zn) two-metal center. Although in all structures of glycosylhydrolase class-13 amylases known so far, the calcium in domain B is coordinated by not more than six protein ligands, in PWA, it is coordinated by seven protein ligands, thus providing a structural rationale for the high binding affinity of the latter enzyme. In addition, 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). Activation of PWA by traces of zinc is, however, superseded by the competitive active site inhibitory effects of this metal. P. woesei has evolved this structural property as a unique evolutionary adaptation most probably to retain full PWA activity in its extremophilic living condition, utilizing zinc as a positive and negative regulator in a concentration-dependent manner. The PWA structures offer a strong base upon which to further engineer properties of PWA that are more conducive to potential applications in industrial processes. In particular, they reveal two metal-binding sites (zinc and calcium) with different functions, which are well suited to optimize the properties of PWA for biotechnological applications.

    ACKNOWLEDGEMENTS

We thank E. Möller (Bayer AG, Leverkusen, Germany) for kindly providing acarbose and D. S. Auld for helpful discussions on the interpretation of metal-binding sites.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The 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 the 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.

Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211339200

2 C. Lu, J. Weizheng, and Y. Yunyan, unpublished data.

    ABBREVIATIONS

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

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