Glycoside Hydrolase Family 66

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• Author: ^^^Ryuichiro Suzuki^^^
• Responsible Curator: ^^^Zui Fujimoto^^^

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Glycoside Hydrolase Family GH66
Clan	none, (β/α)8
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH66.html

Substrate specificities

Glycoside hydrolases of family GH66 include endo-acting dextranases (Dex; EC 3.2.1.11) and cycloisomaltooligosaccharide glucanotransferases (CITase; EC 2.4.1.248). 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 GH49 and GH66.

CITases catalyze intramolecular transglucosylation to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) with degree of polymerization of 7-17 [6]. CITases produce CIs from IG4 and larger IGs [7]. CITase from Bacillus circulans 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 [8]. 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 [9] and chemical rescue studies [1]. GH66 enzymes appear to operate through a classical Koshland retaining mechanism. The k_cat and K_M 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 [2]. Both CITase-T3040 and CITase-598K showed the same K_M value for dextran 40 (0.18 mM) [7]. The k_cat values of CITase-T3040 and CITase-598K against dextran 40 were 3.2 s^-1 and 5.8 s^-1, respectively [7]. Dexs from family GH49 are inverting enzymes.

Catalytic Residues

Catalytic residues of several GH66 enzymes have been identified by mutational and structural studies [1, 7, 9, 10]. 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) [9, 10], Asp340 and Glu412 in Dex from Paenibacillus sp. (PsDex) [1], Asp270 and Glu342 in CITase-T3040 [7, 11], and Asp269 and Glu341 in CITase-598K [7].

Three-dimensional structures

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 [9]. 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 [9]. 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 [11]. 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.

Family Firsts

First stereochemistry determination

CITase-T3040 using ¹H-NMR, ¹³C-NMR, and IR spectra [12].

First catalytic nucleophile identification

Streptococcus mutans SmDex and PsDex by structural study [9] and chemical rescue approach [1], respectively.

First general acid/base residue identification

SmDex and PsDex by structural study [9] and chemical rescue approach [1], respectively.

First 3-D structure

Truncated mutant of SmDex [9] .

References

1. 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. 2012;287(24):19927-35. DOI:10.1074/jbc.M111.339036 |
2. 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. 2012;279(17):3185-91. DOI:10.1111/j.1742-4658.2012.08698.x |
3. Staat RH and Schachtele CF. (1974). Evaluation of dextranase production by the cariogenic bacterium Streptococcus mutans. Infect Immun. 1974;9(2):467-9. DOI:10.1128/iai.9.2.467-469.1974 |
4. 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. 1975;12(6):1415-25. DOI:10.1128/iai.12.6.1415-1425.1975 |
5. Ellis DW and Miller CH. (1977). Extracellular dextran hydrolase from Streptococcus mutans strain 6715. J Dent Res. 1977;56(1):57-69. DOI:10.1177/00220345770560011301 |
6. Funane K, Terasawa K, Mizuno Y, Ono H, Gibu S, Tokashiki T, Kawabata Y, Kim YM, Kimura A, and Kobayashi M. (2008). Isolation of Bacillus and Paenibacillus bacterial strains that produce large molecules of cyclic isomaltooligosaccharides. Biosci Biotechnol Biochem. 2008;72(12):3277-80. DOI:10.1271/bbb.80384 |
7. Suzuki R, Terasawa K, Kimura K, Fujimoto Z, Momma M, Kobayashi M, Kimura A, and Funane K. (2012). Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K. Biochim Biophys Acta. 2012;1824(7):919-24. DOI:10.1016/j.bbapap.2012.04.001 |
8. Funane K, Kawabata Y, Suzuki R, Kim YM, Kang HK, Suzuki N, Fujimoto Z, Kimura A, and Kobayashi M. (2011). Deletion analysis of regions at the C-terminal part of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040. Biochim Biophys Acta. 2011;1814(3):428-34. DOI:10.1016/j.bbapap.2010.12.009 |
9. Suzuki N, Kim YM, Fujimoto Z, Momma M, Okuyama M, Mori H, Funane K, and Kimura A. (2012). Structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66. J Biol Chem. 2012;287(24):19916-26. DOI:10.1074/jbc.M112.342444 |
10. Igarashi T, Morisaki H, Yamamoto A, and Goto N. (2002). An essential amino acid residue for catalytic activity of the dextranase of Streptococcus mutans. Oral Microbiol Immunol. 2002;17(3):193-6. DOI:10.1034/j.1399-302x.2002.170310.x |
11. 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. 2014;289(17):12040-12051. DOI:10.1074/jbc.M114.547992 |

12. Oguma T, Horiuchi T, and Kobayashi M. Novel Cyclic Dextrins, Cycloisomaltooligosaccharides, from Bacillus sp. T-3040 Culture. Biosci Biotechnol Biochem. 1993 57(7):1225-1227. DOI:10.1271/bbb.57.1225