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.

Keywords: Shiro Akabori; enzyme; protein chemistry; protein structure; sugar chain analysis; taka-amylase A.

Fig. 1

Amino acid sequence of TAA. The amino acid sequences were determined by Toda et al. 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. 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.

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.

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.

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. 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.

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.

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.