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Glycoside Hydrolase Family 66
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|Glycoside Hydrolase Family GH66|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolases of family GH66 include endo-acting dextranases (Dex; EC 126.96.36.199) and cycloisomaltooligosaccharide glucanotransferases (CITase; EC 188.8.131.52). Family GH66 enzymes are classified into the following three types: Type I Dexs, Type II Dexs with low CITase activity, and Type III CITases [1, 2].
Dex enzymes hydrolyze α-1,6-linkages of dextran and produce isomaltooligosaccharides (IGs) of varying length. Dex enzymes from oral streptococci have been studied since the 1970s [3, 4, 5]. Dexs are classified into families GH49 and GH66.
CITases catalyze intramolecular transglucosylation to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) with degree of polymerization of 7-17 . CITases produce CIs from IG4 and larger IGs . CITase from Bacillus sp. T-3040 (CITase-T3040) produced CI-8 predominantly from dextran 40, whereas the major product of CITase from Paenibacillus sp. 598K (CITase-598K) was CI-7 [7, 8]. CITases contain a CITase-specific insertion (about 90 residues) inside the catalytic domain. The insertion region is a family 35 carbohydrate-binding module (CBM35) domain . Some Dexs displaying strong dextranolytic activity with low cyclization activity have been discovered [1, 2].
Kinetics and Mechanism
GH66 enzymes are retaining enzymes, as first shown by structural analysis of cyclic dextrins formed by transglycosylation from a-1,6-glucan by Bacillus sp. T-3040 CITase-T3040 . This has been supported by subsequent structural  and chemical rescue studies . GH66 enzymes appear to operate through a classical Koshland retaining mechanism. The kcat and KM values of Dex from Bacteroides thetaiotaomicron VPI-5482 (BtDex) toward dextran T2000 were determined to be 86.7 s-1 and 0.029 mM, respectively . Both CITase-T3040 and CITase-598K showed the same KM value for dextran 40 (0.18 mM) . The kcat values of CITase-T3040 and CITase-598K against dextran 40 were 3.2 s-1 and 5.8 s-1, respectively . Dexs from family GH49 are inverting enzymes.
Catalytic residues of several GH66 enzymes have been identified by mutational and structural studies [1, 7, 10, 11]. The catalytic nucleophile is aspartic acid and the general acid/base is glutamic acid. Asp385 and Glu453 are nucleophile and acid/base catalyst, respectively, in Dex from Streptococcus mutans (SmDex) [10, 11], Asp340 and Glu412 in Dex from Paenibacillus sp. (PsDex) , Asp270 and Glu342 in CITase-T3040 [7, 12], and Asp269 and Glu341 in CITase-598K .
Crystal structures of a truncated mutant of Streptococcus mutans SmDex (lacking the N-terminal 99 and C-terminal 118 residues) have been reported as the first three-dimensional structure of a GH66 enzyme . Three structures, ligand free (PDB ID 3vmn), in complex with IG3 (PDB ID 3vmo), and in complex with 4’,5’-epoxypentyl α-D-glucopyranoside (PDB ID 3vmp), have been solved . The catalytic domain of SmDex is a (β/α)8-barrel fold, accompanied by N-terminal immunoglobulin-like β-sandwich fold and C-terminal β-sandwich structure containing two Greek key motifs. These three domains are the common structural components in GH66 enzymes.
A structure for a GH66 CITase-T3040 (PDB ID 3wnk-3wno) has been reported . CITase-T3040 has a similar domain arrangement to that of SmDex, but a CBM35 domain is inserted into the catalytic module, which assists substrate uptake and production of the dominant cyclooctylisomaltoside (CI-8).
- First stereochemistry determination
- Bacillus sp. T-3040 CITase-T3040 by structural analysis of transglycosylation products using 1H-NMR and 13C-NMR spectroscopy .
- First catalytic nucleophile identification
- Streptococcus mutans SmDex and Paenibacillus sp. PsDex by structural study  and chemical rescue approach , respectively.
- First general acid/base residue identification
- SmDex and PsDex by structural study  and chemical rescue approach , respectively.
- First 3-D structure
- Truncated mutant of SmDex .
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- Hamada S, Mizuno J, Murayama Y, Ooshima Y, and Masuda N. (1975) Effect of dextranase on the extracellular polysaccharide synthesis of Streptococcus mutans; chemical and scanning electron microscopy studies. Infect Immun. 12, 1415-25. DOI:10.1128/IAI.12.6.1415-1425.1975 |
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