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 GH66 contains endo-acting dextranase (Dex; EC 3.2.1.11) and cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248). Dexs hydrolyze α-1,6 linkage of dextran and produce isomaltooligosaccharides (IGs) of varying length. Dexs are classified into GH49 and GH66. In contrast to inverting GH49 enzymes, GH66 enzymes are retaining enzymes. CITases catalyze intramolecular transglucosylation to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) with degree of polymerization of 7-17 [1]. CITases produce CIs from IG4 and larger IGs [2]. CITases from Bacillus circulans T-3040 (CITase-T3040) produced CI-8 predominantly from dextran, whereas the major product of CITase from Paenibacillus sp. 598K (CITase-598K) was CI-7 [2, 3]. CITases contains a CITase-specific insertion (about 90 residues) inside the catalytic domain. The insertion region has been found to be a family 35 carbohydrate-binding module (CBM35) domain that contributes to preference of CI-8 production [3]. Some Dexs displaying strong dextranolytic activity with low cyclization activity have been discovered [4, 5]. The GH66 enzymes are classified into the following three types: (type I) Dexs, (type II) Dexs with low CITase activity, and (Type III) CITases [4, 5].

Kinetics and Mechanism

GH66 enzymes are retaining enzymes, as first shown by structural [6, 7] and chemical rescue studies [4]. 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 [5]. Both CITase-T3040 and CITase-598K showed the same K_M value for dextran 40 (0.18 mM) [2]. The k_cat values of CITase-T3040 and CITase-598K against dextran 40 were 3.2 s^-1 and 5.8 s^-1, respectively [2].

Catalytic Residues

To date, catalytic residues of four GH66 enzymes were identified by mutational and structural studies [2, 4, 7]. In Dex from Streptococcus mutans (SmDex), Asp385 and Glu453 are nucleophile and acid/base catalyst, respectively [7]. In Dex from Paenibacillus sp. (PsDex), Asp340 and Glu412 are nucleophile and acid/base catalyst, respectively [4]. In CITase-T3040, Asp270 and Glu342 are nucleophile and acid/base catalyst, respectively [2]. In CITase-598K, Asp269 and Glu341 are nucleophile and acid/base catalyst, respectively [2].

Three-dimensional structures

The crystal structures of truncated mutant of SmDex (lacking the N-terminal 99 and C-terminal 118 residues) have been reported as the first three-dimensional structure of GH66 enzymes [6, 7]. Three structures, ligand free (PDB code 3VMN), in complex with IG3 (PDB code 3VMO), and in complex with 4’,5’-epoxypentyl-α-D-glucopyranoside (PDB code 3VMP), have been determined. The catalytic domain of the enzyme is a (β/α)₈-barrel fold. The enzyme consists of at least three domains.

Family Firsts

First stereochemistry determination

PsDex by chemical rescue approach [4].

First catalytic nucleophile identification

SmDex and PsDex by structural study [7] and chemical rescue approach [4], respectively.

First general acid/base residue identification

SmDex and PsDex by structural study [7] and chemical rescue approach [4], respectively.

First 3-D structure

Truncated mutant of SmDex [6, 7] .

References

1. 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 |

2. 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 |

3. 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 |

4. 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 |

5. 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 |

6. Suzuki N, Kim YM, Fujimoto Z, Momma M, Kang HK, Funane K, Okuyama M, Mori H, and Kimura A. (2011). Crystallization and preliminary crystallographic analysis of dextranase from Streptococcus mutans. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011;67(Pt 12):1542-4. DOI:10.1107/S1744309111038425 |

7. 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 |