Crystal Structure of Rice {alpha}-Galactosidase Complexed with D-Galactose*

Zui Fujimoto {ddagger}, Satoshi Kaneko §, Mitsuru Momma {ddagger}, Hideyuki Kobayashi § and Hiroshi Mizuno {ddagger} 

From the {ddagger}Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan and the §Biological Function Division, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan

Received for publication, March 5, 2003 , and in revised form, March 20, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
{alpha}-Galactosidases catalyze the hydrolysis of {alpha}-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice {alpha}-galactosidase has been determined at 1.5Å resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of {alpha}-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a ({beta}/{alpha})8-barrel structure, and the C-terminal domain was made up of eight {beta}-strands containing a Greek key motif. The structure was solved as a complex with D-galactose, providing a mode of substrate binding in detail. The D-galactose molecule was found bound in the active site pocket on the C-terminal side of the central {beta}-barrel of the catalytic domain. The D-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice {alpha}-galactosidase with chicken {alpha}-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
{alpha}-Galactosidases ({alpha}-Gals1; E.C. 3.2.1.22 [EC] ) catalyze the hydrolysis of {alpha}-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. {alpha}-Gals are widely distributed in animals, plants, and microorganisms.

In humans, {alpha}-Gal is a lysosomal exoglycosidase that cleaves the terminal {alpha}-galactose residue from glycolipids and glycoproteins. Mutations in the {alpha}-Gal gene cause incomplete degradation of carbohydrates, resulting in Fabry disease (1, 2). In plants, galactomannan is one of the major storage polysaccharides in seeds, and {alpha}-Gal is one of the key enzymes in the degradation of cell wall galactomannan during germination (3, 4). Raffinose and stachyose in beans are known to cause flatulence, and {alpha}-Gal has the potential to alleviate these symptoms (5). In the sugar beet industry, {alpha}-Gal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (6).

We have purified and sequenced several {alpha}-Gals from Mortierella vinacea, Penicillium purpurogenum, Thermus sp. T2, and Oryza sativa L. and have elucidated the substrate specificities of these enzymes using two types of the galactomanno-oligosaccharides, 63-mono-{alpha}-D-galactopyranosyl-{beta}-1,4-mannotriose and 63-mono-{alpha}-D-galactopyranosyl-{beta}-1,4-mannotetraose (7, 8, 9, 10, 11, 12, 13). The results showed that {alpha}-Gals have a diverse preference for substrates. The M. vinacea {alpha}-Gal I and yeast {alpha}-Gals were specific only for 63-mono-{alpha}-D-galactopyranosyl-{beta}-1,4-mannotriose, which has an {alpha}-galactosyl residue (designated the terminal {alpha}-galactosyl residue) linked to the non-reducing end mannose of {beta}-1,4-mannotriose (14, 15). Aspergillis niger {alpha}-Gal and P. purpurogenum {alpha}-Gal, however, showed a preference for 63-mono-{alpha}-D-galactopyranosyl-{beta}-1,4-mannotetraose, which has an {alpha}-galactosyl residue (designated the side-chain {alpha}-galactosyl residue) attached to the inner mannose of {beta}-1,4-mannotetraose (8, 16). M. vinacea {alpha}-Gal II and rice {alpha}-Gal acted on both the terminal {alpha}-galactosyl residue and the side-chain {alpha}-galactosyl residue of the galactomanno-oligosaccharides (9, 13). In addition, M. vinacea {alpha}-Gal II and plant {alpha}-Gals were active on the polymeric substrate galactomannans with high efficiency (9, 17, 18).

To date, the primary structures of over 50 {alpha}-Gals have been deduced from gene or cDNA sequences. Primary structure and hydrophobic cluster analyses have shown that {alpha}-Gals can be classified mainly into the glycoside hydrolase families 4, 27, and 36 (19, 20). {alpha}-Gals from eukaryotes have high amino acid sequence similarities and are basically classified into family 27, whereas bacterial {alpha}-Gals are grouped into families 4 and 36. The hydrolysis mechanism of {alpha}-Gal is known to proceed with retention of the stereochemistry at the anomeric center through the double displacement mechanism, and the nucleophile of the catalytic residue is an aspartic acid that is highly conserved in all known family 27 {alpha}-Gals and other glycosyl hydrolases (21, 22). Recently, the crystal structure of chicken {alpha}-N-acetylgalactosaminidase ({alpha}-NAGal; E.C. 3.2.1.49 [EC] ), which catalyzes the hydrolysis of a terminal {alpha}-N-acetylgalactosamine from glycosylated substrate, has been solved in its free form and in complex with {alpha}-N-acetylgalactosamine (23). This enzyme belongs to family 27 of the glycoside hydrolases and consists of two domains, i.e. an N-terminal domain comprised of a ({beta}/{alpha})8-barrel as a catalytic domain and a C-terminal domain with eight {beta}-strands containing a Greek key motif. The tertiary structure of the catalytic site has provided support for the catalytic mechanism and revealed the mode of substrate binding in {alpha}-NAGal. The structure has also served as a paradigm for constructing models of human {alpha}-NAGal and {alpha}-Gal based on their high amino acid similarities.

In contrast, there has been no report on the three-dimensional structures of the {alpha}-Gals, although a few preliminary crystallization papers have been reported (24, 25). Recently, we have succeeded in carrying out overexpression, purification, and crystallization of the family 27 {alpha}-Gal from rice (O. sativa L. subsp. japonica var. Nipponbare) (13, 26). Rice {alpha}-Gal consists of 362 amino acid residues, and its molecular mass is ~40 kDa. We report here that the crystal structure of rice {alpha}-Gal in complex with D-galactose was determined at 1.5 Å resolution. This structure may be useful in future protein engineering studies and the rational design of {alpha}-Gals for industrial use. We also make structural comparisons of rice {alpha}-Gal with chicken {alpha}-NAGal with a view toward understanding the substrate recognition mechanism in these enzymes.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Crystallization and Data Collection—Rice {alpha}-Gal was purified and crystallized as reported previously (13, 26). The needle- and/or rod-shaped crystals of rice {alpha}-Gal were obtained within 2 weeks by mixing 5 µl of protein solution (15 mg/ml) with 5 µl of reservoir solution (5%2-propanol, 0.1 M ammonium sulfate, 0.1 M acetate buffer, pH4.5, with 5% D-galactose) at 293 K, employing the hanging-drop vapor diffusion method. Diffraction experiments on the native crystals were first carried out at beamline BL6A, Photon Factory, Tsukuba, Japan ({lambda} = 0.978 Å). {alpha}-Gal crystals were flash-frozen in a nitrogen stream at 100 K using 20% glycerol as a cryo-protectant. Diffraction data were collected using a Quantum CCD x-ray detector (Area Detector Systems Corporation, Poway, CA) in 2° oscillation steps over a range of 180°. The native data set was processed and scaled using DPS/MOSFLM (27).

Structure Determination—Heavy atom derivatives of the crystals were prepared by conventional soaking methods for the multiple isomorphous replacement (MIR) method. The heavy atom compounds were dissolved in mother liquor at a final concentration of 10–20 mM. Soaked crystals were left at room temperature for 2–3 h, then soaked in glycerol solution, and then flash-frozen in a nitrogen stream at 100 K. Data sets were collected using the CuK{alpha} x-ray diffractometer and imaging plate detector R-axis IV2+ (Rigaku, Japan). The interpretation of the heavy atom derivative data was initially done using self-vector verification of three-dimensional difference Patterson functions with a CCP4 program package (28). Two mercury positions of the methylmercury chloride derivative were clearly obtained from the Patterson maps. The gold positions of the AuK(CN)2 derivative were then obtained from difference Fourier maps made with single isomorphous replacement phases from the mercury derivative. Two mercury and two gold positions were obtained, and these heavy atom parameters were refined and the phases were computed with program MLPHARE (29) in the CCP4 program package using reflections from 50–1.9 Å resolution. Data collection and heavy atom refinement statistics are shown in Table I. The MIR phase was improved by solvent flattening and density modifications using the program DM (30) in the CCP4 program package. Model building was initially conducted using the auto-modeling program ARP/wARP (31), and the R-factor and the connectivity improved to 19.3 and 98%, respectively, after two trials of 100-cycle calculations. Further model rebuilding was conducted manually with program QUANTA98 (Accelrys). The model was refined with program CNS (32). During the course of refinement, Fo - Fc maps were calculated, and clear densities corresponding to the galactose molecule were found in the active site pocket. Similarly, the bound water molecules were identified. A crystallographic R-factor and an Rfree-factor, which was calculated by setting aside 10% of the reflection data, were improved to 19.0% and 22.9%, respectively. Subsequent refinements were carried out to 1.5 Å resolution using a new native data set taken at the beamline BL41XU at SPring-8, Harima, Japan. Diffraction data were collected using the MAR CCD x-ray detector (MAR Research, Evanston, IL) in 1° oscillation steps over the range of 180°, and the data set was processed and scaled using the programs DENZO and SCALEPACK in the HKL2000 package (33). Six glycerol molecules and a sulfate ion were assigned by calculating the 2Fo - Fc and the Fo - Fc maps. The final R-factor and the Rfree-factor of the model were 16.0% and 17.8%, respectively (Table II). Stereochemistry of the model was analyzed with program PROCHECK (34).


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TABLE I
Data collection statistics of rice {alpha}-galactosidase

Values in parentheses refer to the highest resolution shell.

 

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TABLE II
Structure refinement statistics of rice {alpha}-galactosidase

 


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Overall Structure of Rice {alpha}-Gal—The crystal structure of rice {alpha}-Gal has been determined at 1.5Å resolution by the MIR method using mercury and gold derivatives. Data collection statistics are listed in Table I, and structure refinement statistics are tabulated in Table II. The final model consisted of a single chain of 362 amino acids with a catalytic domain (1–278) and a C-terminal domain (279–362, Fig. 1). The catalytic domain was comprised of a ({beta}/{alpha})8-barrel, which was observed earlier in a triose phosphate isomerase (35) and which now represents a common motif in many glycoside hydrolases. The active site pocket was found on the C-terminal side of the central {beta}-barrel of the catalytic domain where a D-galactose molecule, which was added during crystallization, was identified as a binding ligand. Two disulfide bonds were located near the catalytic pocket. The C-terminal domain was made up of eight {beta}-strands containing a Greek key motif. The interface between the two domains mainly involved hydrophobic interactions. The role of the C-terminal domain is still unknown. In a Ramachandran plot (36), Tyr-20, located on the surface and close to one of the disulfide bonds, was in the generous region, and Asp-216, buried in the catalytic pocket, was in the disallowed region, although electron densities for both residues were clearly observed. The remaining residues were in the most favored region or the additional allowed region.



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FIG. 1.
Stereo view of the ribbon model of rice {alpha}-galactosidase. The bound D-galactose, two catalytic residues, and two disulfide bonds are indicated by ball-and-stick drawings and shown in black, red, and green, respectively. The figure was drawn with the program Raster3d (39, 40).

 

The overall structure was similar to that of chicken {alpha}-NAGal (Protein Data Bank codes 1KTB [PDB] and 1KTC [PDB] ; Ref. 23). Fig. 2 shows the amino acid sequence alignment of rice {alpha}-Gal with chicken {alpha}-NAGal and human {alpha}-Gal. Most of the amino acids comprising the basic secondary structures of the ({beta}/{alpha})8-barrel in the catalytic domain and the Greek key motif in the C-terminal domain are seen to be well conserved. A stereo view of the superimposed C{alpha}-models of rice {alpha}-Gal and chicken {alpha}-NAGal is shown in Fig. 3. Although the length of the {alpha}-Gal molecule is slightly shorter than that of chicken {alpha}-NAGal, largely due to deletions in some loop regions (Fig. 2), the structures of the two enzymes, especially the core region of the ({beta}/{alpha})8-barrel, are basically in good agreement, with root-mean-square differences for C{alpha} atoms of the catalytic domains and the entire two molecules being 1.49 Å and 1.68 Å, respectively.



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FIG. 2.
Amino acid sequence alignment of rice {alpha}-galactosidase with those of human {alpha}-galactosidase and chicken {alpha}-N-acetylgalactosaminidase. The alignment of {alpha}-Gal and {alpha}-NAGal was prepared based on the superposed models. The catalytic residues and the ligand-binding residues are indicated by red and green backgrounds, respectively. The figure was drawn using program ALSCRIPT (41).

 


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FIG. 3.
Stereo view of the C{alpha}–superimposed models of rice {alpha}-galactosidase and chicken {alpha}-N-acetylgalactosaminidase. {alpha}-Gal is shown in orange and {alpha}-NAGal in blue. The two catalytic residues, the disulfide bonds in both enzymes, the D-galactose molecule bound in {alpha}-Gal, and the {alpha}-N-acetylgalactosamine molecule in {alpha}-NAGal are shown.

 

The Active Site and Substrate Binding—Under the crystallization conditions, the addition of D-galactose served to improve resolution of the crystal. The crystal structure of {alpha}-Gal was thus solved as a complex with D-galactose. Fig. 4 shows the Fo - Fc omit map for bound D-galactose and two catalytic aspartates. The density map at 1.5 Å resolution gave clear identification of a chair conformation of the sugar ring and the orientation of the hydroxyl groups. At the anomeric C1 carbon, multi-conformational electron densities corresponding to both {alpha}-anomeric and {beta}-anomeric O1 hydroxyl groups were clearly observed, indicating that the D-galactose molecules was bound as a mixture of two anomeric forms. From the intensity of the densities, the {alpha}-anomer and {beta}-anomer were estimated to be present in the ratio of 0.3 and 0.7, respectively, which is similar to the ratio of the natural abundance of 0.36/0.64 for the solution state of galactopyranose. Furthermore, the B-factors of these atoms calculated to be almost the same when the occupancy of these atoms were set at this ratio. The {alpha}-anomeric hydroxyl group projected outwards, whereas the {beta}-anomeric hydroxyl group pointed to the side of the active pocket. In an enzymatic reactions involving the two anomers, the enzyme selectively binds to the {alpha}-galacto-oligosaccharide, because the enzyme cannot bind to the {beta}-galacto-oligosaccharide whose {beta}-anomeric oxygen would be pointed toward the bottom of the catalytic pocket. But, if the monomeric galactose molecule were in high concentration, it is possible that it may act as a competitor of the substrate.



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FIG. 4.
Stereoview of the Fo - Fc omit electron density map for the bound D-galactose and two catalytic residues contoured at 3.5 {sigma}

 

Fig. 5A shows the structure of the {alpha}-Gal/galactose complex around the catalytic pocket. The galactose molecule is seen buried in the catalytic pocket with the {alpha}-anomeric O1 hydroxyl group open to the surface. Catalytic residues found in rice {alpha}-Gal were two aspartic acids, i.e. Asp-130 located at the end of strand {beta}4, and Asp-185 after strand {beta}6. Asp-130 was hydrogen bonded to the {beta}-anomeric O1 hydroxyl oxygen and was located within hydrogen-bonding distance of the O5 atom of the galactose molecule. Another catalytic residue, Asp-185, was hydrogen bonded to the {alpha}-anomeric O1 hydroxyl and O2 hydroxyl oxygen atoms. As proposed previously (23), catalysis by this enzyme seems to involve a double-displacement mechanism. In rice {alpha}-Gal, Asp-185 works as a catalytic acid/base, and Asp-130 acts as a nucleophile inasmuch as Asp-130 is located close to the anomeric carbon C1 atom of the galactose (2.5 Å), and Asp-185 is near the {alpha}-anomeric hydroxyl O1 atom, which corresponds to the oxygen atom in the {alpha}-1,6-glycosidic linkage to substrate. The distance between two carboxyl groups was 6.5Å, which is close to the standard distance of 5.5Å in other normal retaining glycosyl hydrolases (37), considering that the galactose molecule is accommodated over a distance of two residues.



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FIG. 5.
Stereo view around the catalytic pocket. A, rice {alpha}-galactosidase. Bound sugar, two catalytic residues, surrounding residues, and disulfide bonds are indicated by ball-and-stick drawings and are shown in black, red, white, and green, respectively. Coordinating hydrogen bonds are shown in broken green lines. B, stereo superimposed models of rice {alpha}-galactosidase and chicken {alpha}-N-acetylgalactosaminidase. {alpha}-Gal is shown in orange, and {alpha}-NAGal is shown in green.

 

Besides the catalytic residues, other residues had hydrophilic and hydrophobic contacts to the ligand. Arg-181, Trp-164, and Cys-162, as well as Asp-185 formed hydrogen bonds with the O2 oxygen atom of galactose. Lys-128 hydrogen bonded to the two hydroxyl oxygen O3 and O4 atoms. Asp-51 had a hydrogen bond to the hydroxyl oxygen O4 atom, and Asp-52 hydrogen bonded to the O6 atom. In addition, hydrophobic contacts were observed for residues Trp-16, Cys-101, and Met-217. The aromatic indole ring of Trp-16 did not face the sugar ring but rather toward a sort of flat surface formed by the O3, C3, C4, C5, C6, and O6 atoms of the bound galactose in a manner which is often seen in galactose-binding proteins and lectins (38). Cys-101 formed one of the two disulfide bridges with Cys-132. A total of 11 residues were involved in galactose binding. A previous study has shown that this enzyme is completely inhibited by p-chloromercuribenzoic acid is and strongly inhibited by Hg2+ and Ag2+ (13). Because Cys-162 is one of three free cysteine residues in the catalytic pocket, these metal reagents may attack the cysteine and interfere with substrate binding in the catalytic site.

Specificity against Monomeric Galactose—The catalytic pocket of rice {alpha}-Gal was similar to that of chicken {alpha}-NAGal. Fig. 5B shows the structure around the bound ligand of rice {alpha}-Gal superimposed onto that of chicken {alpha}-NAGal. In both of these, most of the surrounding residues were well conserved, and any difference in the two could be seen only in a single loop between strand {beta}5 and helix {alpha}5. The loop of {alpha}-NAGal was longer by six amino acids than that of {alpha}-Gal, making a short helical structure that was not conserved in rice {alpha}-Gal (Fig. 3). In {alpha}-NAGal, Tyr-176 and Arg-197 sandwiched the whole N-acetyl moiety, and Ser-172 and Ala-175 coordinated with the carbonyl oxygen atom and the methyl group, respectively. Garman et al. (23) has designated this loop as "N-acetyl recognition loop." In the case of rice {alpha}-Gal, Trp-164 and Cys-162 in this loop replaced Ala-175 and Ser-172 of {alpha}-NAGal, filling up the cavity for the N-acetyl group so that alternatively this loop could also be referred to as "N-acetyl blocking loop" or "substrate discriminating loop". The pocket for {alpha}-NAGal is longer than that for {alpha}-Gal, providing space necessary for the N-acetyl group. It is possible that this longer loop evolved by changes in the amino acid composition to accommodate these different substrates.

The amino acid sequence homology of human {alpha}-Gal with respect to chicken {alpha}-NAGal is 50% compared with 37% for rice {alpha}-Gal (Fig. 2). As far as the substrate discriminating loop is concerned, human {alpha}-Gal has a longer loop resembling that of chicken {alpha}-NAGal, such that the cavity used for accommodating the N-acetyl group could be occupied by leucine and glutamic acid in human {alpha}-Gal in place of the corresponding residues Ala-175 and Ser-172 of {alpha}-NAGal, as discussed by Garman et al. (23). The leucine, glutamic acid, and neighboring amino acid residues might be considered well conserved in the two {alpha}-Gals if Leu-161 is inserted at the end of the fifth {beta}-strand of rice {alpha}-Gal. However, this single insertion of Leu-161 would cause a drastic change in the positions and orientations of Cys-162, Glu-163, and Trp-164 as well as a disruption of the third disulfide bridge. In turn, Trp-164 or Cys-162 would have to assume a new role in order to recognize the galactose substrate moiety. Among members of the {alpha}-Gal family, most of the enzymes from bacteria, yeast, and plant have this single amino acid insertion before the substrate discriminating loop compared with the enzymes in animal (13). Considering these facts, the single insertion might represent the point at which branching off occurred during the evolution of the {alpha}-Gals.

Specificity against Galacto-oligosaccharides—The rice {alpha}-Gal, as well as the other eukaryotic {alpha}-Gals, can accommodate both terminal and side-chain {alpha}-galactosyl residues. The crystal structure of the galactose complex showed that the {alpha}-anomeric oxygen atom of the bound galactose molecule projected directly toward the solvent area, leaving enough space in the catalytic pocket so that the bound {alpha}-galactosyl residue in the catalytic pocket could be linked to either the terminal or the inner sugar of the manno-oligosaccharide through an {alpha}-1,6-glycosidic bond. It is still unclear whether the enzyme recognizes the other sugars of the substrate besides the {alpha}-galactosyl moiety. But, at the surface, Trp-16 and Trp-164 contributed partly to a build up of the catalytic pocket, whereas Tyr-20 and His-18 were localized on the surface near the catalytic pocket. Because aromatic residues are often used in recognizing the sugar moieties in enzymes and lectins (38), the above residues might be considered candidates for substrate recognition.

It has been shown that some bacterial and yeast {alpha}-Gals can cleave only terminal {alpha}-galactosyl or only side-chained {alpha}-galactosyl residues (8, 14, 15, 16). For examples, M. vinacea {alpha}-Gal I and Saccharomyces cerevisiae {alpha}-Gal, which act on only the terminal {alpha}-galactosyl residues, have a 20-amino acid insertion before the helix {alpha}6, whereas P. purpurogenum {alpha}-Gal, which acts on only the side-chained {alpha}-galactosyl residues, has a 30-amino acid insertion before helix {alpha}4. Because the sequences of the core {beta}/{alpha}-barrel are highly conserved among various {alpha}-Gals, these insertions could cause the extended loops to bulge out and possibly obstruct the catalytic pocket, thereby limiting accessibility to the side-chained or terminal {alpha}-galactosyl residues. Alternatively, they may cause more subsites against {alpha}-galactosyl oligosaccharides to be formed, resulting in multiple specificities against the {alpha}-galactosyl moiety. As we still lack sufficient data to model the inserted loops, the three-dimensional structures of the other types of {alpha}-Gals are necessary to clarify these differences in substrate specificities.


    FOOTNOTES
 
* This work was performed with the approval of the Photon Factory, the High Energy Accelerator Research Organization, Japan (proposal number 2000G294) and SPring-8 (proposal number 2002A0130-NL1-np) and supported in part by Rice Genome Project Grants PR-2106 and PR-2206, Ministry of Agriculture, Forestry, and Fisheries, Japan. 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. Back

To whom correspondence should be addressed: Dept. of Biochemistry, National Inst. of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan, Tel.: 81-29-838-7014; Fax: 81-29-838-7408; E-mail: mizuno{at}affrc.go.jp.

1 The abbreviations used are: {alpha}-Gal, {alpha}-galactosidases; {alpha}-NAGal, {alpha}-N-acetylgalactosaminidase; MIR, multiple isomorphous replacement. Back


    ACKNOWLEDGMENTS
 
We thank Drs. M. Suzuki, and N. Igarashi for their help in the data collection at the Photon Factory and Drs. M. Kawamoto and H. Sakai at SPring-8 BL41XU.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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