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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 188.8.131.52) and cycloisomaltooligosaccharide glucanotransferases (CITase; EC 184.108.40.206). 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 one CBM35 domain is inserted into the catalytic module, and assist the substrate uptake and dominant CI-8 production.
- First stereochemistry determination
- Bacillus sp. T-3040 CITase-T3040 using 1H-NMR, 13C-NMR, and IR spectra .
- 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 .
- Kim YM, Kiso Y, Muraki T, Kang MS, Nakai H, Saburi W, Lang W, Kang HK, Okuyama M, Mori H, Suzuki R, Funane K, Suzuki N, Momma M, Fujimoto Z, Oguma T, Kobayashi M, Kim D, and Kimura A. (2012) Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach. J Biol Chem. 287, 19927-35. DOI:10.1074/jbc.M111.339036 |
- Kim YM, Yamamoto E, Kang MS, Nakai H, Saburi W, Okuyama M, Mori H, Funane K, Momma M, Fujimoto Z, Kobayashi M, Kim D, and Kimura A. (2012) Bacteroides thetaiotaomicron VPI-5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions. FEBS J. 279, 3185-91. DOI:10.1111/j.1742-4658.2012.08698.x |
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- Suzuki N, Fujimoto Z, Kim YM, Momma M, Kishine N, Suzuki R, Suzuki S, Kitamura S, Kobayashi M, Kimura A, and Funane K. (2014) Structural elucidation of the cyclization mechanism of α-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase. J Biol Chem. 289, 12040-51. DOI:10.1074/jbc.M114.547992 |