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). Dex enzymes hydrolyze α-1,6 linkages of dextran and produce isomaltooligosaccharides (IGs) of varying length. Dex enzymes from oral streptococci have been analyzed since 1970s [1, 2, 3]. Dexs are classified into GH49 and GH66. In contrast to inverting GH49 enzymes, GH66 enzymes show retaining enzymatic properties. CITases catalyze intramolecular transglucosylation to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) with degree of polymerization of 7-17 [4]. CITases produce CIs from IG4 and larger IGs [5]. 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 [5, 6]. 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 [6]. Some Dexs displaying strong dextranolytic activity with low cyclization activity have been discovered [7, 8]. The GH66 enzymes are classified into the following three types: (Type I) Dexs, (Type II) Dexs with low CITase activity, and (Type III) CITases [7, 8].

Kinetics and Mechanism

GH66 enzymes are retaining enzymes, as first shown by structural [9] and chemical rescue studies [7]. 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 [8]. Both CITase-T3040 and CITase-598K showed the same K_M value for dextran 40 (0.18 mM) [5]. The k_cat values of CITase-T3040 and CITase-598K against dextran 40 were 3.2 s^-1 and 5.8 s^-1, respectively [5].

Catalytic Residues

To date, catalytic residues of four GH66 enzymes have been identified by mutational and structural studies [5, 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) [7], Asp270 and Glu342 in CITase-T3040 [5, 11], and Asp269 and Glu341 in CITase-598K [5].

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 [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 determined [9]. The catalytic domain of the enzyme 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.

The second structure for GH66 has been reported for CITase-T3040 (PDB ID 3wnk-3wno)[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

SmDex and PsDex by structural study [9] and chemical rescue approach [7], respectively.

First general acid/base residue identification

SmDex and PsDex by structural study [9] and chemical rescue approach [7], respectively.

First 3-D structure

Truncated mutant of SmDex [9] .

References

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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