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The Concept of the Α-Amylase Family

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The Concept of the Α-Amylase Family International Symposium on Extremophiles and Their Applications 2005 The concept of the !-amylase family: a rational tool for interconverting glucanohydrolases/glucanotransferases, and their specificities Takashi Kuriki Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan Email: [email protected] Abstract Neopullulanase catalyzes the hydrolysis of !-1,4- and !-1,6-glucosidic linkages, as well as transglycosylation to form !-1,4- and !-1,6-glucosidic linkages. Based on the series of experimental results using the 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 !-amylase family. Mutational and structural analyses provided the conclusive proof that one active center of neopullulanase participate in all four reactions. We have been trying to interconvert glucanohydrolases/glucanotransferases, and their specificities and create tailor-made industrially useful enzymes based on the concept of the !-amylase family. We engineered Thermus amylomaltase to essentially erase hydrolytic activity and created perfect 4-!-glucanotransferase for the industrial production of cycloamylose. Keywords; !-amylase family, neopullulanase, catalytic mechanism, catalytic machinery, protein engineering 1. Introduction All enzymes known to date have individual EC number 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 the “key-lock” hypothesis 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 similar folding pattern is often seen in different enzymes which have clearly distinct functions [2]. The concept of !-amylase family [3] was proposed independently from the classification of carbohydrate-active enzymes based on amino acid sequence similarities [4, for updated form http://afmb.cnrs-mrs.fr/CAZY/]. In 1989, we had already pointed out the common catalytic machinery among !-amylase (EC 3.2.1.1), cyclomaltodextrin glucanotransferase (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 substrate-binding residues that bind glucosyl residues adjacent to the scissile linkage in the substrate even though each enzyme has different function, based on the experimental results for neopullulanase [5] (Fig. 1). 50 International Symposium on Extremophiles and Their Applications 2005 Enzyme Origin Region1 Region2 Region3 Region4 !-A myl ase As per gil lus or yzae 11 7 DVVANH 20 2 GLRI DTVKH 23 0 EVLD 29 2 FVENH D CG Tas e Ba cil lus ma cer ans 13 5 DFAPNH 22 5 GIRF DAVKH 25 8 EWFL 32 4 FIDNH D Pu llu lan ase Kl ebs iel la aer ogen es 60 0 DVVYNH 67 1 GFRF DLMGY 70 4 EGWD 82 7 YVSKH D Is oam yla se Ps eud omo nas am ylod era mos a 29 2 DVVYNH 37 1 GFRF DLASV 43 5 EPWA 50 5 FIDVH D Br anc hin g e nzy me Es che ric hia co li 33 5 DWVPGH 40 1 ALRV DAVAS 45 8 EEST 52 1 LPLSH D Ne opu llu lan ase Ba cil lus st ear othe rmo phi lus 24 2 DAVFNH 32 4 GWRL DVANE 35 7 EIWH 41 9 LLGSH D Am ylo pul lul ana se Th erm oan aer oba cter et han oli cus 48 7 DGVFNH 59 3 GWRL DVANE 62 6 ELWG 69 8 LLGSH D !-G luc osi das e Sa cch aro myc es carl sbe rge nes is 10 6 DLVINH 21 0 GFRI DTAGL 27 6 EVAH 34 4 YIENH D Cy clo dex tri nas e Th erm oan aer oba cter et han oli cus 23 8 DAVFNH 32 1 GWRL DVANE 35 4 EVWH 41 6 LIGSH D Ol igo -1, 6-g luc osid ase Ba c il lus ce reu s 98 DLVVNH 19 5 GFRM DVINF 25 5 EMPG 32 4 YWNNH D De xtr an glu cos idas e St rep toc occ us muta ns 98 DLVVNH 19 0 GFRM DVIDM 23 6 ETWG 30 8 FWNNH D Am ylo mal tas e St rep toc occ us pneu mon iae 22 4 DMWAND 29 1 IVRI DHFRG 33 2 EELG 39 1 YTGTH D Gl yco gen de bra nchi ng en zym e Ho mo sap ien s 29 8 DVVYNH 50 4 GVRL DNCHS 53 4 ELFT 60 3 MDITH D Am ylo suc ras e Ne iss eri a p oly sacc har ea 19 0 DFIFNH 29 0 IL RM DAVAF 33 6 EAIV 39 6 YVRSH D Tr eha los e-6 -ph osph ate hy dro las e Es che ric hia co li 10 0 DMVFNH 19 6 GLRL DVVNL 25 1 EMSS 32 0 FWCNH D Ma lto oli gos ylt reha los e h ydr ola se Ar thr oba cto r sp . 20 2 DVVYNH 26 3 GLRL DAVHA 30 4 ESDL 39 5 CSQNH D Ma lto oli gos ylt reha los e s ynt has e Ar thr oba cto r sp . 87 DIVPNH 23 3 GLRI DHPDG 26 4 EKIL 47 8 TLSTH D Tr eha los e s ynt hase Pi mel oba cto r sp. 108 DFVMNH 20 6 GF RL DAVPY 25 2 EANQ 32 2 F L RNH D Fig. 1 Enzymes belonging to !-amylase family and four highly conserved regions. Invariable three catalytic sites are highlighted by inversion. Numbering of the amino acid sequences of the enzymes starts at the amino-terminal amino acid of each mature enzyme. The study on neopullulanase was the key to open the door for the formulation of the concept of the !-amylase 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 !-1,4- and !-1,6-glucosidic linkages [8], as well as transglycosylation to form !-1,4- and !-1,6- glucosidic 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 !-amylase family [3]. We describe here the concept of the !-amylase family focusing attention on our work. Other important contributions to establish the concept have been also described in excellent reviews [10, 11, 12]. We defined !-amylase family as one enzyme family that satisfy the following requirements: the !-amylase family enzymes; (i) act on !-glucosidic linkages; (ii) hydrolyze !-glucosidic linkages to produce !-anomeric mono- and oligo-saccharides or form !- glucosidic 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 Taka-amylase A [3]. We did not include the ("/!)8-barrel structure which is commonly seen in !-amylase family enzymes as one of our original definitions of !-amylase family. From the viewpoints of the convergent evolution of the ancestral proteins and the chemical reactions, we think that there should be many enzymes that catalyze the same reaction even though they have totally different structures [2]. Indeed, the xylanases from Cellulomonas fimi and Streptomyces lividans are composed of ("/!)8-barrel structure although the xylanase from Bacillus circulans is composed of "-jelly roll (http://afmb.cnrs- mrs.fr/CAZY/). We think that the importance may lie in the local geometrical arrangements of catalytic and substrate-binding residues, but not in the whole structure of the protein. Whole structure may reflect the stable topological motif of the protein in water and the evolutional relationship. In fact, recent study revealed the existence of !-amylases, 51 International Symposium on Extremophiles and Their Applications 2005 amylopullulanases (EC 3.2.1.1/41), and 4-!-glucanotransrerases (2.4.1.25) in the proteins having ("/!)7-barrel structure [13]. By the classification of carbohydrate-active enzymes based on amino acid sequence similarities (http://afmb.cnrs-mrs.fr/CAZY/), these enzymes are classified into glycoside hydrolase families 57 (GH-57), although the enzymes belonging to !-amylase family were in clan GH-H covering three glycoside hydrolase families 13, 70, and 77 (GH-13, 70, and 77) [14]. This is also a fundamentally different point between the concept of the !-amylase family and the classification of carbohydrate-active enzymes based on amino acid sequence similarities (http://afmb.cnrs-mrs.fr/CAZY/). In order to explain the concept of the !-amylase family, we schematically represented the relationship of specificities for the target linkage and reaction of the enzymes typically belonging to the !-amylase family [2, 3, 6, 15] (Fig. 2). Hydrolysis of !-1,4-glicosidic linkage is typically catalyzed by !-amylase. Pullulanase and isoamylase hydrolyze !-1,6 linkage [16, 17]. Amylomaltase catalyzes transglycosylation to form !-1,4-glucosidic linkage. Branching enzyme catalyzes transglycosylation to form !-1,6 linkage [18-21]. These four reactions and classification of these enzymes have been clearly distinguished. Each of the four reactions is representatively catalyzed by one of four individual types of enzymes. However, some exceptional examples have been also reported.
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