The Concept of the .ALPHA.-Amylase Family: A Rational Tool for Interconverting Glucanohydrolases/Glucanotransferases, and Their Specificities

Abstract

All enzymes known to date have individual EC numbers based on the recommendation of the International Union of Biochemistry and Molecular Biology (IUBMB). This enzyme classification is based on two factors, the reaction catalyzed and the substrate specificity. This is consistent with thekeylockhypothesis postulated by Emil Fischer in 1884. It has been estimated that more than 25,000 enzymes exist in nature. However, Chothia 1) speculated that the number of basic topological motifs of protein is most likely limited to around one thousand. Therefore, it is quite natural that a similar folding pattern is often seen in different enzymes which have clearly distinct functions. 2) The concept of the αamylase family 3) was proposed independently from the classification of carbohydrateactive enzymes based on amino acid sequence similarities 4) (for updated form http:afmb.cnrsmrs.frCAZY). In 1989, we had already pointed out the common catalytic machinery among αamylase (EC 3.2.1.1), cyclomaltodextrin glu-canotransferase (EC 2.4.1.19), pullulanase (EC 3.2.1.41), isoamylase (EC 3.2.1.68) and neopullulanase, and the existence of four highly conserved regions which contains all the catalytic residues and the substratebinding residues that bind glucosyl residues adjacent to the scissile linkage in the substrate even though each enzyme has a different function, based on the experimental results for neopullula-nase 5) (Fig. 1). The study on neopullulanase was the key to open the door for the formulation of the concept of the αamylase family. 6) We found a new enzyme, neopullulanase (EC 3.2.1.135) from Bacillus stearothermophilus, 7) and showed that it catalyzes the hydrolysis of α1,4 and α1,6 glucosidic linkages, 8) as well as transglycosylation to form α1,4 and α1,6glucosidic linkages. 3) The replacement of several amino acid residues that constitute the active center of the neopullulanase showed that one active center of the enzyme participated in all four reactions described above. 9) Pointing out the same catalytic machinery and the common catalytic mechanism of the enzymes that catalyze these four reactions, we proposed and defined a general idea for the αamylase family. 3) We describe here the concept of the αamylase family focusing attention on our work. Other important contributions to establish the concept have been also described in excellent reviews. 1012) We defined the αamylase family as one enzyme family that satisfy the following requirements: the αamylase family enzymes (i) act on αglucosidic linkages; (ii) hy-drolyze αglucosidic linkages to produce αanomeric mono and oligosaccharides or form αglucosidic linkages by transglycosylations; (iii) have four highly conserved regions in their primary sequences which contain all the catalytic and most of the important substrate binding sites; and (iv) have Asp, Glu and Asp residues as catalytic sites corresponding to Asp206, Glu230 and Asp297 of Takaamylase A. 3) We did not include the (β α)8barrel structure which is commonly seen in αamylase family enzymes as one of our original definitions of the Abstract: We found a new enzyme, neopullulanase (EC 3.2.1.135), and showed that it catalyzes the hydrolysis of α-1,4-and α-1,6-glucosidic linkages, as well as transglycosylation to form α-1,4-and α-1,6-glucosidic linkages. Based on the series of experimental results using neopullulanase, we pointed out the same catalytic machinery and the common catalytic mechanism of the enzymes that catalyze these four reactions, and thus, proposed and defined the concept of the α-amylase family. Mutational analyses provided the evidence that one active center of neopullulanase participates in all four reactions; the hydrolysis of α-1,4-and α-1,6-glucosidic linkages and transglycosylation to form α-1,4-and α-1,6-glucosidic linkages. Structural analyses provided the conclusive proof that one active center of neopullulanase participates in all four reactions. We have been trying to interconvert glucanohydrolasesglucanotransferases, and their specificities and create tailor-made industrially useful enzymes based on the concept of the α-amylase family. Based on the concept, we engineered Thermus amylomaltase to essentially erase hydrolytic activity and created perfect 4-α-glucanotransferase for the industrial production of cycloamylose. The concept of the α-amylase family is demonstrated here again as a rational tool for interconverting glucanohydrolasesglucanotransferases, and their specificities.