Glycoside Hydrolase Family 68

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• Author: ^^^Tirso Pons^^^ and ^^^Wim Van den Ende^^^
• Responsible Curator: ^^^Wim Van den Ende^^^

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Glycoside Hydrolase Family GH68
Clan	GH-J
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH68.html

Substrate specificities

Glycoside hydrolase family GH68 contains enzymes that hydrolyze fructose containing polysaccharides such as levansucrase (sucrose:2,6-β-D-fructan 6-β-D-fructosyltransferase; EC 2.4.1.10); β-fructofuranosidase (EC 3.2.1.26); and inulosucrase (EC 2.4.1.9)

Kinetics and Mechanism

Family GH68 enzymes are retaining enzymes, as first shown by Koshland and Stein by performing the reaction in ¹⁸O-labeled water and determining the ¹⁸O content of the products [1]. The levansucrases from Bacillus subtilis, Gluconacetobacter diazotrophicus, and Streptococcus salivarius follows a ping-pong mechanism [2, 3, 4, 5]. At low sucrose concentrations levansucrase functions as a hydrolase with water as acceptor, whereas at higher substrate concentrations it adds fructosyl units to a growing levan chain [2].

Catalytic Residues

Retaining glycosidases catalyze hydrolysis in two steps involving a covalent glycosyl enzyme intermediate. The two invariant residues, responsible for the catalytic reaction in family GH68 enzymes, have first been identified experimentally in bacterial levansucrases as an aspartate located close to the N-terminus acting as the catalytic nucleophile and a glutamate acting as the general acid/base [6, 7]. In addition, a conserved aspartate residue in the "Arg-Asp-Pro (RDP) motif" stabilize the transition state [7].

Three-dimensional structures

Currently, only two different three dimensional structures of family GH68 enzymes have been solved so far. The first crystal structure was reported for the bacterial levansucrase (SacB) from Bacillus subtilis subsp. subtilis str. 168 [6]. The second one corresponds to levansucrase (LdsA) from Gluconacetobacter diazotrophicus SRT4 [8].

Family Firsts

First stereochemistry determination

Cite some reference here, with a short (1-2 sentence) explanation [1].

First catalytic nucleophile identification

Cite some reference here, with a short (1-2 sentence) explanation [9].

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation [10].

First 3-D structure

Cite some reference here, with a short (1-2 sentence) explanation [3].

References

You can even cite books using just the ISBN [11]. References that are not in PubMed can be typed in by hand [9].

1. KOSHLAND DE Jr and STEIN SS. (1954). Correlation of bond breaking with enzyme specificity; cleavage point of invertase. J Biol Chem. 1954;208(1):139-48. | Google Books | Open Library
2. Chambert R, Treboul G, and Dedonder R. (1974). Kinetic studies of levansucrase of Bacillus subtilis. Eur J Biochem. 1974;41(2):285-300. DOI:10.1111/j.1432-1033.1974.tb03269.x |
3. Chambert R and Gonzy-Tréboul G. (1976). Levansucrase of Bacillus subtilis: kinetic and thermodynamic aspects of transfructosylation processes. Eur J Biochem. 1976;62(1):55-64. DOI:10.1111/j.1432-1033.1976.tb10097.x |
4. Hernandez L, Arrieta J, Menendez C, Vazquez R, Coego A, Suarez V, Selman G, Petit-Glatron MF, and Chambert R. (1995). Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochem J. 1995;309 ( Pt 1)(Pt 1):113-8. DOI:10.1042/bj3090113 |
5. Song DD and Jacques NA. (1999). Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975. Biochem J. 1999;341 ( Pt 2)(Pt 2):285-91. | Google Books | Open Library
6. Meng G and Fütterer K. (2003). Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Biol. 2003;10(11):935-41. DOI:10.1038/nsb974 |
7. Yanase H, Maeda M, Hagiwara E, Yagi H, Taniguchi K, and Okamoto K. (2002). Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase. J Biochem. 2002;132(4):565-72. DOI:10.1093/oxfordjournals.jbchem.a003258 |
8. Martínez-Fleites C, Ortíz-Lombardía M, Pons T, Tarbouriech N, Taylor EJ, Arrieta JG, Hernández L, and Davies GJ. (2005). Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus. Biochem J. 2005;390(Pt 1):19-27. DOI:10.1042/BJ20050324 |

9. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006