Crystals of taka-amylase A, a cornerstone of protein chemistry in Japan

Abstract

In 1935, Shiro Akabori began research on the preparation of taka-amylase A with a purity suitable for chemical research, with the intention of elucidating the chemical nature of the enzyme. He succeeded in developing a method to efficiently obtain crystallized taka-amylase A from Aspergillus oryzae. Using crystallized taka-amylase A as the starting material, a series of studies were conducted to determine its amino acid composition and sequence, sugar chain structure, and three-dimensional structure. Based on these results, the molecular structure and catalytic mechanism of taka-amylase A were elucidated. The scientific achievements from research on taka-amylase A significantly enhanced Japan’s capabilities in protein research, represented by the fact that taka-amylase A was the first amylase in the world for which both chemical and crystallographic structures were elucidated.

1. Introduction

In 1922, Shiro Akabori entered the Faculty of Science, Tohoku University, Japan, where he studied organic chemistry under the guidance of Professor Rikō Majima in 1924. Akabori studied the aromatic components of soy sauce. After he learned that aromatic compounds are produced during fermentation, he studied fermentation chemistry and the enzymes that control fermentation. In 1927, as a graduate student, he attended a lecture on “Recent Issues in Protein Research” by Takaoki Sasaki,¹⁾ which led him to research enzymes.²⁾

Akabori was conferred his Doctor of Science degree in 1931 for “Studies on Amino Acids and Their Derivatives”. He went on to study at the laboratory of Professor Ernst Waldschmidt-Leitz at the German Technical University in Prague in 1932. In this laboratory, Akabori examined a conventional method for proline quantification and demonstrated that this method quantifies hydroxyproline, not proline.

During that period, the predominant view on enzymes in Europe was the so-called “carrier theory”, wherein enzyme activity was attributed to active groups adsorbed onto proteins or colloids, rather than to the proteins themselves. In contrast, in the United States (U.S.), James B. Sumner succeeded in crystallizing jack bean urease in 1926 and announced it was a protein.³⁾ However, this view was not widely accepted. In 1933, while Akabori was studying in Prague, a dispute arose between Waldschmidt-Leitz and Sumner regarding the chemical nature of the enzyme.^4),5)

As Akabori was unconvinced by the claims of Waldschmidt-Leitz et al., he spent three months at Professor John H. Northrop’s laboratory at the Rockefeller Institute in the U.S. on his way home from Prague. Northrop et al. had successfully crystallized pepsin,⁶⁾ trypsin,⁷⁾ and chymotrypsin.⁸⁾ Akabori learned from Dr.Moses Kunitz about the process of purification and activation of chymotrypsinogen and trypsinogen and was convinced that enzymes are proteins.

He returned to Japan in 1935 and became an associate professor under Professor Majima, who moved to Osaka University as a professor at the Faculty of Science, and immediately began research on enzyme chemistry.

2. Crystallization of taka-amylase

2.1. Selecting taka-amylase as a research target.

Although enzymes extracted from animal organs were mainly studied in Europe and the U.S., they were difficult to obtain in Japan. In contrast, the enzymes produced by Japanese koji mold (Aspergillus oryzae) are generally more stable than animal-derived enzymes, which is an immense advantage for chemical studies.²⁾ Jokichi Takamine developed the world-renowned digestive agent Takadiastase,⁹⁾ which was derived from culturing koji mold and wheat bran and has been sold commercially since the late 19th century by Parke-Davis and Sankyo corporations. Takadiastase, manufactured in large quantities at Sankyo Corporation, contains taka-amylase as its main constituent. Taka-amylase efficiently catalyzes the hydrolysis of starch and has a high potential for industrial-scale production of glucose from various sources. Akabori developed a keen interest in Takamine himself and taka-amylase, and he aimed to resolve scientific questions related to taka-amylase, including its chemical structure and catalytic mechanism.^10),11) Thus, Akabori selected taka-amylase as a research target, analyzed its physicochemical properties, and developed isolation and crystallization methods to obtain taka-amylase of high purity for chemical studies.

2.2. Beginning of chemical studies on taka-amylase.

Chemical studies of enzymes begin with first obtaining the enzyme in a pure state. To this end, Akabori et al. conducted studies on the crystallization of taka-amylase.

First, they treated taka-amylase with trypsin or papain, which are protein-digesting enzymes; however, no change in amylase activity was observed. In contrast, positive results were obtained for taka-amylase in Molisch’s test, suggesting that taka-amylase contained sugar.¹²⁾

Taka-amylase was not digested using papain; however, the precipitate formed when these two were mixed showed strong amylase activity. This suggested the possibility of purifying taka-amylase via fractionated precipitation.^13),14)

After examining various protein precipitants, Shigeya Hayashi found that rivanol (2-ethoxy-6,9-diaminoacridine monolactate) was particularly effective as a fractional precipitant of taka-amylase. A precipitate was produced upon the addition of a rivanol solution to obtain partially purified taka-amylase. After dissolving the precipitate in acetate buffer, rivanol was easily removed by adsorption onto the bleached soil. The mother solution was treated with ammonium sulfate, and the resulting precipitate was dialyzed to obtain a clear, colorless enzyme solution with high specific activity.¹⁵⁾ However, the taka-amylase obtained in these preparations did not crystallize.

2.3. Analysis of activation and inactivation mechanism of taka-amylase.

Kinetic analysis of the auto-reactivation of acid-inactivated taka-amylase showed that its recovery rate did not depend on enzyme concentration or solvent viscosity, indicating that reactivation was a first-order reaction. When acid-inactivated taka-amylase was dialyzed, the hypothetical active groups and carriers of this enzyme did not dissociate and separate. These results indicated that taka-amylase is neither activated by the combination of the active groups and the carrier nor inactivated by the dissociation of the active groups and the carrier in the acid solution. Accumulated evidence contradicted the carrier theory for taka-amylase.¹⁶⁾

2.4. Success in crystallization of taka-amylase A.

After World War II, Akabori and his colleagues resumed their research on the crystallization of taka-amylase.

In 1951, Akabori et al. prepared crude taka-amylase by culturing A. oryzae in a synthetic culture medium and adding calcium acetate solution to the resulting sample. The proteolytic enzymes present in the culture medium were inactivated using heat treatment, and the precipitate was removed. The solution was dialyzed against water, and ammonium sulfate was added to obtain crude taka-amylase as a precipitate. The precipitate was dissolved in cold water and dialyzed, and rivanol was added to precipitate taka-amylase. After the removal of rivanol, taka-amylase in the dissolved solution was precipitated with acetone, and the precipitate was re-dissolved in a calcium acetate solution. After adding acetone, the resulting enzyme solution was stored in an icebox to generate fine cubic crystals of taka-amylase. After 16 years, taka-amylase was finally crystallized. The crystallized taka-amylase showed strong biuret and Millon reactions,¹⁷⁾ which specifically detect peptide bonds and phenol groups, providing evidence of its protein nature.

In 1953, Hiroshi Okazaki reported the existence of low-molecular-weight amylase in “Takadiastase Sankyo”¹⁸⁾; Takadiastase manufactured by Sankyo Corporation; therefore, the amylase crystallized by Akabori et al. is called “taka-amylase A” (hereafter abbreviated as TAA).

In 1954, TAA crystals were obtained, with a high yield (30%), from commercially available Takadiastase Sankyo by introducing rivanol precipitation to the purification method.¹⁹⁾ Crystallized TAA exhibited all the properties of a protein and appeared to be a calcium salt. This preparation was electrophoretically pure, although it exhibited weak digestive activity in horse serum and hemoglobin.

Based on these results, chemical studies on the amino acid composition, amino acid sequence, sugar chain structure, and three-dimensional structure of TAA have been conducted by many researchers in different fields, and efforts have been made to improve its purity for each purpose.

3. Amino acid composition of TAA

When Akabori began to analyze the chemical structure of TAA in the 1950s, the technology applicable to protein analysis was limited. Furthermore, the molecular weight of TAA was more than 50,000, suggesting that TAA was probably the largest protein studied for structural analysis at that time.

After acid-hydrolysis of crystallized TAA, Akabori et al. determined the amino acid composition of TAA in 1954.²⁰⁾ Amino acids in the hydrolysates were individually analyzed using colorimetric and enzymatic methods. Additionally, the amino acids were quantified after separation using starch and ion exchange columns. Using a combination of these results, the amino acid composition of TAA was deduced, and the molecular weight of TAA was estimated to be 54,000 ± 700.

In 1963, Hiroko Toda and Akabori developed a new method for preparing TAA from Takadiastase extracts using diethylaminoethyl cellulose (DEAE-C) chromatography.²¹⁾ TAA in the Takadiastase extracts was sequentially purified using batchwise DEAE-C chromatography, acetone precipitation with calcium acetate, and gradient elution DEAE-C chromatography. The TAA obtained was reduced and carboxymethylated (RCM), and hydrolyzed, and its amino acid composition was measured using an automated analyzer by the Kozo Narita and Toda group.²²⁾ The resulting data were similar to the amino acid composition calculated from the amino acid sequence finally determined by Toda et al. in 1982²³⁾; however, several amino acids showed slightly lower values than the finally determined values. In 1966, Narita further purified the DEAE-C preparation and reported more definitive amino acid analysis results.²⁴⁾

4. Amino- and carboxy-terminal analysis of TAA

In 1955, Akabori and Tokuji Ikenaka deduced the amino (N)-terminal residue of TAA using Sanger’s method.^25),26) After reacting with dinitrofluorobenzene (DFB) and hydrolyzing with hydrochloric acid, dinitrophenyl (DNP)-amino acids were separated on a silica gel column and identified as DNP-Ala.²⁵⁾ The molecular weight of TAA was estimated to be 52,500 via quantification of DNP-Ala.

At that time, no universal and convenient method was available for identifying carboxy (C)-terminal amino acids. Akabori et al. developed a new method using hydrazine, which was produced in large quantities as rocket fuel during World War II and was no longer required after the war.²⁷⁾ Anhydrous hydrazine decomposes proteins into amino acid hydrazides, except for the C-terminal amino acid which remains intact. The amino acid hydrazides were converted into dibenzal compounds and extracted using an organic solvent. The C-terminal amino acids in the aqueous phase were identified using paper or silica gel chromatography. An advantage of this technique is that proteins are soluble in anhydrous hydrazine. Hydrazinolysis was established as a C-terminal analysis method after several revisions.^28)-30)

Ikenaka determined the C-terminal amino acids of crystallized TAA using this method and observed three amino acids, Ser, Gly, and Ala, in 1956, suggesting that TAA comprised a branched polypeptide chain.³¹⁾ This erroneous result was probably obtained due to low-molecular weight-peptide impurities generated by the action of proteolytic enzymes that faintly contaminated the crystallized TAA, as pointed out in Ikenaka’s reinvestigation study conducted with Narita and Hironori Murakami in 1966.²⁴⁾ They identified the C-terminal residue as Ser by carboxypeptidase digestion and hydrazinolysis using the purified DEAE-C preparation of TAA described above. Collectively, TAA was confirmed to be a linear polypeptide with N-terminal Ala and C-terminal Ser residues.

5. Amino acid sequence analysis of TAA

Narita and Akabori analyzed the N-terminal sequence of TAA using the Sanger sequencing method.³²⁾ After reacting with DFB, DNP-TAA was partially hydrolyzed, and the resulting DNP-peptides were purified and analyzed in 1959.³³⁾ However, the proposed sequences did not match those determined in 1982.²³⁾ We acknowledge the difficulty of sequence determination in the 1950s, before the introduction of the Edman method, which utilizes a stepwise coupling/cleavage reaction with phenylisothiocyanate followed by identification of phenylthiohydantoin (PTH)-amino acids that are converted from the released thiocarbamoyl derivatives.³⁴⁾

In 1965, the Narita and Toda group reported that one Cys was present in the free form and masked in the structure of TAA, whereas the other eight Cys residues formed disulfide bonds.²²⁾ Narita and Mitsutaro Akao labeled the sulfhydryl group of the masked Cys and digested it with trypsin and pepsin. The labeled Cys peptide was isolated, and its amino acid sequence was determined using the manual Edman and carboxypeptidase methods.³⁵⁾

Narita proceeded with the sequence analysis of TAA, mainly using its tryptic peptides. Although one-third of the tryptic peptides showed low solubility, a large C-terminal fragment of RCM-TAA was obtained in a high yield. This fragment was digested with thermolysin and separated using chromatography, and its 66-residue sequence was determined using the manual Edman method in 1975.³⁶⁾ In 1980, the Toda and Narita group reported an N-terminal 29-residue sequence of TAA using an automated liquid-phase protein sequencing technique.³⁷⁾

In 1982, the Toda and Narita group reported the amino acid sequence of the 478-residue TAA (Fig. 1).²³⁾ This sequence was determined using 10 cyanogen bromide (CNBr)-cleaved peptides of RCM-TAA, which were purified using serial chromatography. Met-containing peptides that constituted the connecting CNBr-peptides were purified from the tryptic digests of maleylated RCM-TAA, and the digests were demaleylated, digested, and separated. The amino acid sequences of these peptides were determined using automated liquid- andsolid-phase sequencing and manual Edman degradation. PTH-amino acids were identified using high-performance liquid chromatography (HPLC) or thin-layer chromatography. The main reasons for the long time taken to complete the whole sequencing were as follows: i) TAA was too large to systematically obtain sequence data in the 1950s and the 1960s; ii) a large portion of the fragmented peptides were insoluble or had low solubility; and iii) the early TAA preparations contained significant amounts of impurities.

Fig. 1

Amino acid sequence of TAA. The amino acid sequences were determined by Toda et al.²³⁾ The amino acids shown beneath the sequence were in disagreement with the genomic DNA sequence of α-amylase 1 (amy1) of A. oryzae reported by Wirsel et al.³⁸⁾ Additionally, one insertion (W between residues 384 and 385) and one deletion (D476) were observed. One nearly identical gene (amy3) is present in this fungus, which has two replacements at Q35R and F151L compared to those in amy1 (underlined). C30–C38, C150–C164, C240–C283, and C439–C474 form disulfide linkages. C227 is present in its free form, and N197 is glycosylated (double underlined). Two catalytic residues, D206 and E230, are boxed. Single-letter codes for amino acids are used.

Genomic and complementary DNA sequences of TAA were determined in 1989 using the aforementioned amino acid sequences. Three nearly identical α-amylase genes are present in A. oryzae, namely amy1, amy2, and amy3; amy1 and amy2 have the same translated sequence, but amy2 is not transcribed.³⁸⁾ Several groups have determined amy1 and/or amy3 gene sequences,^39)-41) which are now curated and available in a public database (UniProt) under the accession numbers P0C1B3 (amy1 and amy2) and P0C1B4 (amy3). The amino acid sequence reported by Toda et al. corresponded to amy1, in which one insertion, one deletion, and eight conflicts were observed (Fig. 1). However, enormous difficulties occurred during the 478-residue sequencing process. We would like to acknowledge the tremendous effort and patience required to complete the TAA sequencing performed by Narita, Toda, and their colleagues.

When the TAA sequence analysis was completed, the number of protein sequence determination studies rapidly increased. Subsequently, Hunkapiller and Hood developed a new sequencer called “the gas-phase sequencer,” coupled with a sensitive PTH-amino acid-identifying HPLC system.⁴²⁾ With the introduction of this instrument, protein sequencing has become a popular and convenient technique for researchers. Further improvements enabled one-picomole-level protein/peptide sequencing using Edman chemistry. DNA sequencing technology has also made dramatic progress, and protein sequencing has often been utilized since the middle of the 1980s to determine partial amino acid sequences for designing DNA probes to screen clones carrying protein-coding sequences.

6. Structural analysis of sugar chains on TAA

Chemical structure analysis of sugar chains linked to proteins is difficult due to their structural heterogeneity and the need for multiple complicated chemical reactions to determine the sugar composition, sugar linkage order, position, and anomers. The sugar chain of TAA is a rare case in which the structure was determined solely by chemical methods.

In 1937, Akabori estimated that TAA contained sugar chains using the Molisch reaction¹²⁾; however, the possibility of contamination of purified TAA with sugar could not be ruled out because protein purification was difficult at that time. Following the crystallization of TAA in 1951,^17),19) Hidesaburo Hanafusa et al. performed monosaccharide composition analysis in 1955 and showed that TAA contained two moles of hexosamine, eight moles of mannose, and one mole of xylose.⁴³⁾ They also found that TAA derived from A. oryzae grown in different cultures had a sugar chain content of approximately 1/10. To analyze the sugar chain structure, Motoaki Anai et al. obtained glycopeptides from pronase-digested fragments of TAA using an anion exchange resin in 1966.⁴⁴⁾ Haruki Yamaguchi, Ikenaka, and Yoshio Matsushima further purified the glycopeptide with a uniform sugar chain structure containing two moles of N-acetylglucosamine and six moles of mannose residues in 1969.⁴⁵⁾ The glycopeptide was subjected to Smith degradation, which acts on the diol structure,⁴⁶⁾ methylation analysis, which is used to determine the positions of sugar linkages,⁴⁷⁾ and partial acetolysis, which provides information on the branching structures.⁴⁸⁾ These products were analyzed using gas chromatography, which was available then, to estimate the positions of the glycan linkages.^49),50) Matsushima and Ikuo Yamashina’s groups determined the α and β anomers of each sugar linkage using the purified anomer-specific N-acetyl-β-glucosaminidase, α-mannosidase, and β-mannosidase.^51)-53) Yamaguchi et al. determined the sugar chain structures of glycopeptides isolated from TAA (Fig. 2).⁵⁰⁾ Determination of the structure of this sugar chain took approximately six years and was performed using a combination of complicated chemical techniques. Subsequently, the amino acid sequence linked to the sugar chain in TAA was determined by Satoko Isemura and Ikenaka to be Asn-Glu-Trp-Tyr-Asp-Trp-Val-Gly-Ser-Leu-Val-Ser-Asn(sugar chain)-Tyr-Ser-Ile-Asp-Gly-Leu-Arg.⁵⁴⁾ The results validated the hypothesis proposed at that time that the sugar chains were covalently linked to the Asn residue in the consensus sequence (Asn-X-Ser/Thr, where X was an amino acid residue other than Pro).

Fig. 2

The sugar chain structure of the TAA-glycopeptide. The composition, sequence, linkage positions, and anomers of the constituent sugars were determined using chemical methods.

Around that time, more than 10 sugar structures of glycoproteins, besides TAA, were reported.⁵⁵⁾ Although many of the sugar chain structures reported are now known to contain errors, the accurate structure of the sugar chain on TAA was determined at that time.⁵⁰⁾ This is largely because of the homogeneous structure of the glycopeptide in TAA, which can be prepared on a gram scale for structural analysis. Sugar chain structures are generally heterogeneous; however, the relatively uniform sugar chain structure of TAA is thought to be due to the action of carbohydrate hydrolases expressed in A. oryzae. Few glycoproteins have uniform sugar chains, as in this case.⁵⁶⁾

Four years after the report on the sugar chain structure of TAA, Akira Kobata et al. reported a sugar chain with the same structure as that of TAA in avian (chicken) albumin, and they demonstrated the commonalities in the sugar chain structures of glycoproteins across species.⁵⁷⁾ A review on glycoprotein sugar chains published in 1975 emphasized that the sugar chain structural analysis of TAA contributed to determining the general structure of sugar chains on glycoproteins.⁵⁸⁾

7. Developments in sugar structural analysis and glycobiology

For TAAs with low carbohydrate content,⁴³⁾ Sumihiro Hase et al. showed in 1984 that only one N-acetylglucosamine residue was linked to TAA.⁵⁹⁾ Because the enzymatic activities of TAAs with different sugar chain structures do not change, the function of sugar chains was unknown, and sugar chain structures did not attract the attention of many life science researchers. In addition, the complexity of structural analysis methods hampered efforts to extensively analyze the entire structure of sugar chains. Therefore, developing a simplified and sensitive method for analyzing sugar chain structures was essential. In the 1970s, sugar chain structural analysis methods were developed, including the tritium labeling method developed by Kobata et al.⁶⁰⁾ and the fluorescent labeling method developed by Hase et al.⁶¹⁾ (Fig. 3). The fluorescent labeling method was developed by the group that determined the sugar chain structure of TAA. This method is widely used because it allows chromatographic separation of sugar chains at the pmol level. Fluorescent-labeled sugar chains have also been used to identify and analyze carbohydrate-related enzymes with low expression levels. In the 1990s, Naoyuki Taniguchi et al. identified numerous glycosyltransferases responsible for synthesizing sugar chains in glycoproteins using fluorescent-labeled sugar chains.⁶²⁾ These sugar chains are essential for specific ligand-receptor interactions and the regulation of cell-to-cell recognition, signal transduction, immunity, and carcinogenesis.⁶³⁾ Thus, the field of glycobiology in Japan can be considered to have begun with the structural analysis of TAA sugar chains and the developments in this field continue to occur to this day.

Fig. 3

Tritium-labeling and fluorescent-labeling methods used for sugar chain structural analysis. Glycans modified at reducing ends can be detected with high sensitivity by radioactivity or fluorescence analysis.

8. Three-dimensional structure of TAA

In 1951, Masao Kakudo, an X-ray crystallographer at Osaka University, heard of the successful crystallization of TAA by Akabori and became highly interested in the enzyme crystals. When Kakudo visited Akabori’s laboratory, Ikenaka showed him a large, square, pale-yellow crystal, which surprised him and stimulated a desire for research further. Protein crystallography had not yet begun in Japan at that time. In January 1958, Kakudo was studying at the Brooklyn Institute of Technology, where he had an opportunity to hear the first lecture on the successful crystallographic analysis of myoglobin at 6 Å resolution by Dr. John C. Kendrew, who had come from the Royal Institution in London.

In 1958, Akabori established the Institute for Protein Research at Osaka University. As the first director of the institute, Akabori established the Division of Protein Physical Structure in 1959 to initiate protein crystallography in Japan and invited Kakudo to become the professor-in-charge. Kakudo prepared and collected the analytical equipment for protein crystal structure analysis with Yoshio Sasada and Tsunehiro Takano. In 1959, a three-circle diffractometer, which is essential for protein crystallography, was installed. In 1967, an NEC NEAC 2200 computer was donated to Osaka University. The Osaka University team of X-ray crystallography, led by Tamaichi Ashida, established programs available to all researchers working on crystal structure analyses of small molecules. The computer programs required for each protein structure analysis were custom-written by researchers working on each protein. In 1968, an automatic four-circle X-ray diffractometer designed by Tatsuo Ueki in collaboration with Rigaku Electric Co. Ltd. was introduced for high-speed intensity acquisition of protein samples.

Kakudo and his colleagues elucidated the three-dimensional structure of bonito cardiac ferrocytochrome c (molecular weight, approximately 12,000), whose single crystals had been under preparation for X-ray crystallography since the late 1960s.⁶⁴⁾ The group completed the three-dimensional structural analysis of bonito cardiac ferrocytochrome c at 6 Å resolution in 1969, 4 Å resolution in 1971,⁶⁵⁾ and 2.3 Å resolution in 1973. Moreover, the group succeeded in building a molecular skeletal model.⁶⁶⁾ This was the first atomic-resolution structural analysis of a protein in Japan and the first structural analysis of ferrocytochrome c worldwide.

In parallel, Kakudo et al. investigated the conditions for preparing single crystals of TAA for X-ray diffraction experiments. TAA was purified from Takadiastase Sankyo using the method described by Akabori et al.,¹⁹⁾ followed by DEAE-Sephadex column chromatography. Single crystals obtained using ammonium sulfate as the precipitant were suitable for X-ray diffraction analysis, whereas those obtained by acetone precipitation were not.

One millimeter-sized crystals of TAA with 478 amino acid residues were used for diffraction data collection. At that time, TAA was the highest molecular weight target for crystallographic analysis. The space group of the TAA crystal was P2₁, with cell dimensions of a = 91.9, b = 133.3, c = 94.3 Å, and β = 102.7°. Three TAA molecules were present in the asymmetric unit.

Yoshiki Matsuura, Masami Kusunoki, and their colleagues collected TAA diffraction data at 6 Å resolution, which was obtained using Cu-K α radiation on a Rigaku four-circle diffractometer equipped with a rotating anode X-ray generator (40 kV; 200 mA).⁶⁷⁾ This equipment was first manufactured in Japan and greatly improved the rate of intensity-data collection. Data from five TAA crystals were averaged to obtain 5,400 independent reflections, resulting in 6 Å resolution data for the native crystal. Seven heavy-atom derivatives, HgCl₂, UO₂(NO₃)₂, AgNO₃, K₂PdCl₄, KAu(CN)₂, K₂PtCl₄, and K₂Pt(CN)₄, were used for phase determination, and a multiple heavy-atom isomorphous substitution method and an anomalous dispersion effect were combined to obtain phase data.

The apparent shape of the TAA molecule was an ellipsoid with approximate dimensions of 80 × 45 × 35 Å. Twelve different heavy-atom-binding sites were observed, some of which were occupied by two or more types of heavy atoms.

Three significant humps in the TAA molecule were observed at common positions in all three molecules.⁶⁷⁾ The different electron-density maps obtained for the inhibitors, which were 6′-deoxy-6′-iodomaltose (DIM) and 6′-deoxy-6′-iodomaltotriose (DIMT) derivatives, showed almost the same features. The four inhibitor-binding sites were located within a limited area of the molecular surface. One of the inhibitor sites was situated in a hollow, extending over the heavy-atom sites. The positions of certain amino acid residues were estimated based on heavy-atom binding modes.⁶⁸⁾ The approximate shape of the TAA was determined using the analytical image with a resolution of 6 Å. In addition, the substrate-binding and active sites of this enzyme, along with the amino acid residues present at these sites, were deduced from the analysis of the hollow and heavy-atom binding positions on TAA.

Subsequently, Matsuura and his colleagues started analyzing the three-dimensional structure of TAA at 3 Å resolution to obtain detailed structural information.⁶⁹⁾ The molecular model of TAA was constructed using a Richards box, wherein electron-density maps were drawn on transparent plastic sheets. Diffraction data were obtained using 29 native TAA crystals and 89 heavy-atom derivative crystals with approximately 39,600 independent reflections in the 6–3 Å resolution range, which improved the quality of the electron-density map. A three-dimensional protein model was built by matching the amino acid sequences determined by Toda et al.²³⁾

The coordinates of approximately half of the peptide backbone and side-chain atoms for each residue were read using a string with a pendulum, whereas those of the remaining atoms were calculated using a stereochemical computer program, assuming that each residue of the TAA molecule had a standard bond length and angle, as determined by high-resolution X-ray analysis for 20 different amino acids. TAA was the first amylase in the world whose amino acid sequence and three-dimensional structure were determined. The structural analysis of TAA pioneered protein crystallography in Japan, which in turn facilitated the elucidation of the efficient catalytic mechanism underlying starch degradation, thereby increasing its applicability and utility at the forefront of the food industry. It has been recognized as Chemical Heritage No. 051 by the Chemical Society of Japan. The coordinates, except those of the carbohydrates, were deposited in the Protein Data Bank (PDB ID: 2TAA) in 1982.^70),71)

9. Catalytic mechanism of taka-amylase A

The TAA molecule consists of two domains.⁷⁰⁾ The first 380 residues from the N-terminus constitute domain A, and the rest constitute domain B, where domain A contains a (β/α)₈ super-secondary structure. Domain B has an eight-stranded, antiparallel sandwich structure. Electron-density maps showed a carbohydrate moiety linked to Asn197, located on the opposite side of the catalytic cleft. One Ca²⁺, which is strongly bound to and commonly required for α-amylase, is not directly involved in catalytic activity but contributes to maintaining the structure of the catalytic cleft (Fig. 4).

Fig. 4

The overall structure of TAA. The right side of the molecule consists of an N-terminal (β/α)₈-barrel structure, and the left side is composed of an 8-stranded antiparallel β-sheet sandwich. Glu230 and Asp206 are catalytic residues, with the former serving as a proton donor (acid/base) and the latter as a nucleophile.⁷⁴⁾ CA indicates strongly bound Ca²⁺, supporting the three-dimensional structure of the catalytic cleft. The carbohydrate moiety is linked to the side chain of Asn197. N and C indicate the N- and C-termini, respectively. The pairs of numbers indicate the positions of the respective cystine residues that form disulfide bonds. Helical ribbons and arrows show α-helices and β-strands, respectively. The figure was prepared using PyMol (Schrӧdinger) based on the PDB ID 2TAA.⁷⁰⁾

In domain A, a large cleft was observed around the carboxyl end of the parallel barrel. Differential Fourier map analysis revealed that maltose molecules generated by the hydrolysis of maltotriose, a poor substrate of amylase, and the inhibitor DIMT bind in the vicinity of Glu230, suggesting that this cleft is the catalytic site of this enzyme. The amino acid residues presumed to comprise the substrate-binding site in this cleft are highly conserved in porcine pancreatic α-amylase and TAA.²³⁾

Among the conserved amino acid residues, Glu230, Asp297, and Asp206 are candidate catalytic amino acids. At pH 5.5, the optimum pH for the hydrolysis reaction catalyzed by TAA, the carboxyl group of Glu230 in a hydrophobic environment is thought to be non-ionized, whereas the carboxyl group of Asp297 in a hydrophilic environment is thought to be ionized. Matsuura et al. hypothesized that these two residues cooperatively hydrolyze the glycosidic bond via an acid-base catalytic mechanism.⁷⁰⁾

After accumulating data on amino acid residue modifications and crystal structure analyses of α-amylases belonging to the same family, Asp206, Asp297, and Glu230 were concluded to be essential residues for amylase activity.^70),72),73) Asp206 and Glu230 are catalytic residues, serving as the nucleophile and the proton donor (acid/base), respectively.⁷⁴⁾ The two catalytic amino acid residues, Asp206 and Glu230, are boxed in Fig. 1. The configuration of the glycosidic bond was retained after hydrolysis (Fig. 5).

Fig. 5

Possible catalytic mechanism leading to the hydrolysis of the α-glycosidic bond by TAA. Glycosidic oxygen is protonated by the proton donor Glu230. Nucleophilic Asp206 attacks the C1 atom of glucose on the non-reducing side of the glycosidic oxygen, forming a covalent enzyme intermediate. Subsequently, the intermediate is hydrolyzed by a water molecule, which is the second nucleophilic substitution step. The α-configuration of the glucose C1 atom is retained.

Elucidating the reaction mechanism of α-amylases is important for industrial applications. For this reason, attempts have been made to analyze the structure of the complex of α-amylase and its substrate, the environment of the catalytic site, and the hydrogen bonding network using computer simulation.^75),76) By combining these data with the three-dimensional structures of TAA and other amylases, the molecular mechanisms of enzymatic activity were proposed (Fig. 5). The report on the structure of TAA (PDB ID: 2TAA) is currently the most cited PDB structure in the Journal of Biochemistry.⁷⁷⁾

The authors would like to express their deep respect for Kakudo and his colleagues, who not only established protein crystallography in Japan but also greatly contributed to the development of structural biology in Japan, and the training of many people involved in life science research based on X-ray crystallography.

The crystallization of TAA brought together many researchers to elucidate the chemical nature of this enzyme. In the process of this research, many talented individuals were nurtured under the guidance of Dr.Shiro Akabori (Fig. 6), and together with senior researchers in Japan, they accomplished historical scientific achievements through their studies on TAA.

Fig. 6

Shiro Akabori (October 20, 1900–November 3, 1992), DSc (Tohoku Imperial University, 1931). Shiro Akabori was the President of Osaka University, Professor Emeritus at Osaka University, President of RIKEN, and was awarded the Grand Cordon of the Order of the Sacred Treasure. He was elected as a member of the Japan Academy, the German Academy of Natural Sciences Leopoldina, and the USSR Academy of Sciences. He was also an honorary member of the American Society of Biological Chemists.

Acknowledgments

The authors would like to express their gratitude to Professors Emeritus Toshio Takagi and Sumihiro Hase of Osaka University for their comments on the background of TAA research. The authors would like to thank Associate Professor Kazuhiro Matsunaga of the Tekijuku Memorial Center of Osaka University for his efforts to introduce materials showing the relationship between Jokichi Takamine and Tekijuku and the librarians at Osaka University Library for their assistance in collecting academic information.

Notes

Edited by Yoshinori FUJIYOSHI, M.J.A.

Correspondence should be addressed to: S. Aimoto, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan (e-mail: [e-mail]).

Footnotes

This paper commemorates the 100th anniversary of this journal and introduces the following paper previously published in this journal. Akabori, S., Hagihara, B. and Ikenaka, T. (1951) Purification and crystallization of taka-amylase. Proc. Jpn. Acad. 27 (7), 350-351 ( https://doi.org/10.2183/pjab1945.27.350).

References

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Related Articles

• Akabori, S., Hagihara, B. and Ikenaka, T. (1951) Purification and crystallization of taka-amylase. Proc. Jpn. Acad. 27 (7), 350-351.

Appendices

[From Proc. Jpn. Acad., Vol. 27 No. 7, pp. 350-351 (1951)]

Non-standard abbreviation list

CNBr

cyanogen bromide

DEAE-C

diethylaminoethyl cellulose

DFB

dinitrofluorobenzene

DIM

6′-deoxy-6′-iodomaltose

DIMT

6′-deoxy-6′-iodomaltotriose

DNP

dinitrophenyl

HPLC

high-performance liquid chromatography

PTH

phenylthiohydantoin

RCM

reduced and carboxymethylated

TAA

taka-amylase A

Profile

Saburo Aimoto was born in Yamaguchi Prefecture, Japan, in 1947 and graduated from the Faculty of Science, Osaka University in 1970. He received his Doctor of Science degree in 1977 from Osaka University under the supervision of Prof. Yoshiharu Izumi. He joined Dr. Yasutsugu Shimonishi's lab at the Institute for Protein Research, Osaka University, and worked as an assistant professor (1972-1987). During this time, he worked as a postdoctoral fellow in Prof. Gopinath Kartha's lab at the Roswell Park Memorial Institute (1978-1979), and in Prof. Frederic M. Richards's lab at the Department of Molecular Biophysics and Biochemistry, Yale University (1979-1980). Returning to the Institute for Protein Research, he worked as an associate professor (1987-1993) and a professor (1994-2011). He majored in organic chemistry, especially in synthetic protein chemistry. He has made significant contributions to initiating current innovations in protein synthesis chemistry. He was appointed Director of the Institute for Protein Research in 2008 until 2010 and served as an executive vice president of Osaka University from 2011 to 2015. He moved to the Protein Research Foundation in 2015 and became President from 2016 to 2021. He received the Japanese Peptide Society Award in 2007, the Xiaoyu Hu Memorial Award in 2010 from the Chinese International Peptide Symposium, and the Chemical Society of Japan Award 2010.

Naoto Minamino was born in Osaka in 1953. He received his B.S., M.S., and Ph.D. from Osaka University in 1976, 1978, and 1983, respectively, with Professors Hisayuki Matsuo and Kozo Narita as actual and formal supervisors at the Institute for Protein Research. He started his academic career at Nagasaki University and Miyazaki Medical College. In 1985, he received a JSPS Fellowship for Research Abroad and worked for 2 years in the U.S.-Japan Biomedical Research Laboratory of Professor Akira Arimura, Tulane University. He returned to Miyazaki Medical College as an assistant professor in 1987 and then transferred to the National Cerebral and Cardiovascular Center Research Institute as a laboratory chief in 1989. He worked as a department director from 2002, and then a director of the Omics Research Center from 2015 to his retirement. His research is focused on identifying biologically active peptides and elucidating their physiological roles. He has discovered neo-endorphins, neuromedins, natriuretic peptides, and neuro-endocrine regulatory peptides, among others. He initiated a peptidome project for cataloging naturally occurring peptides to identify new peptides from 1999 and multi-omics analyses to identify diagnostic biomarkers from 2010. He received the Young Investigator Award of the Japanese Biochemical Society in 1989 and the Japanese Peptide Society Award in 2015.

Takeshi Ishimizu was born in Osaka in 1971. He received his B.S. in 1993 and Ph.D. in 1998 from Osaka University. He was involved in enzyme research at the Institute for Protein Research, Osaka University, as a graduate student under the supervision of Prof. Fumio Sakiyama. After spending a year at Pennsylvania State University (Prof. Teh-hui Kao) as a JSPS Research Fellow, he joined the research group of Prof. Sumihiro Hase at the Graduate School of Science, Osaka University, as an assistant professor in 1999. He moved to the College of Life Sciences, Ritsumeikan University as an associate professor in 2012. He was promoted to professor in the same university in 2019. He was awarded the Japanese Biochemical Society's Young Investigator Award in 2010. He has been engaged in basic research on plant carbohydrate-active enzymes and discovered novel enzymes, such as glycoside hydrolases acting on plant $N$-glycans, glycosyltransferases involved in the biosynthesis of plant cell wall polysaccharide pectin, and glycosyltransferases involved in the biosynthesis of flavonoid glycoside apiin.

Masami Kusunoki was born in Osaka Prefecture, Japan, in 1953, and graduated from the Faculty of Science, Osaka University, in 1975. He received his Doctor of Science degree in 1980 from Osaka University under the supervision of Prof. Masao Kakudo in the Institute for Protein Research. The title of his doctoral thesis was “X-ray Structure Analysis of Taka-Amylase A at 3.0 Å Resolution”. He started his career as an assistant professor in Prof. Kakudo's lab at the Institute for Protein Research (1980-1995). He then worked as an associate professor in Prof. Tomitake Tsukihara's Lab at the same institute (1995-2008). He moved to the University of Yamanashi as a professor (2008-2018) and is now a professor emeritus at the University of Yamanashi. His research career has focused mainly on the X-ray analysis of various enzymes and proteins from microorganisms, plants, and animals, performed in collaboration with many laboratories in Japan. He was involved in the early stages of Protein Data Bank (PDB) activities in Japan at the Institute for Protein Research, including the start of local data processing in Japan, with strong technical support from the Research Collaboratory for Structural Bioinformatics (RCSB) of the PDB.