New to the CAZy classification? Read this first.
Want to learn more about CAZypedia? Read the CAZypedia 10th anniversary article in Glycobiology.
Glycoside Hydrolase Family 68
Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.
|Glycoside Hydrolase Family GH68|
|Active site residues||known|
|CAZy DB link|
Glycoside hydrolase family 68 enzymes include levansucrase (sucrose:2,6-β-D-fructan 6-β-D-fructosyltransferase; EC 18.104.22.168), β-fructofuranosidase (EC 22.214.171.124), and inulosucrase (EC 126.96.36.199). All these enzymes use sucrose as their preferential donor substrate. Many of them can create very long levan-type fructans (catalyzed by levansucrases) or inulin-type of fructans (catalyzed by inulosucrases), as well as fructooligosacharides (FOS). However, some GH68 enzymes can also use fructan as donor substrate (in the abscence of sucrose or at a high fructan/sucrose ratio).
Kinetics and Mechanism
Family GH68 enzymes as well as those included in GH32 are retaining enzymes . The levansucrases from Bacillus subtilis, Gluconacetobacter diazotrophicus, and Streptococcus salivarius catalyze transfructosylation via a ping-pong mechanism involving the formation of a transient fructosyl-enzyme intermediate [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 variety of acceptors including glucose, fructan and sucrose . Bacterial levansucrases, whatever their origin, catalyze all these reactions but with different efficiency.
GH68 retaining enzymes 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" stabilizes the transition state [5, 7, 8]. The three equivalent acidic residues have been mutated in a β-fructofuranosidase from Arthrobacter globiformis IFO 3062 , and in a levansucrase and a inulosucrase from Lactobacillus reuteri 121 .
Currently, six different three dimensional structures of family GH68 enzymes have been solved. The first crystal structure was reported for the bacterial levansucrase (SacB) from Bacillus subtilis subsp. subtilis str. 168 . The second one corresponds to levansucrase (LdsA) from Gluconacetobacter diazotrophicus SRT4 , the third corresponds to SacB from Bacillus megaterium , the fourth is an inulosucrase (InuJ) from Lactobacillus johnsonii NCC533  and the fifth one corresponds to beta-fructofuranosidase (ArFFase) from Arthrobacter sp. K-1 . More recently, the three dimensional structure of a levansucrase from Erwinia amylovora has been characterized as well . These structures display a 5-fold β-propeller topology, and therefore GH families 68 and 32 have been combined in clan GH-J. On the other hand, a structural relationship of the catalytic core exists to family GH68 and family GH43, as predicted by detailed sequence analysis .
- First stereochemistry determination
- Bacillus subtilis levansucrase .
- First catalytic nucleophile identification
- Bacillus subtilis levansucrase .
- First general acid/base residue identification
- Zymomonas mobilis levansucrase .
- First stabilizing transition state residue identification
- Gluconacetobacter diazotrophicus levansucrase .
- First prediction of a common beta-propeller catalytic domain in GH68 / clan GH-J
- Gluconacetobacter diazotrophicus levansucrase [17, 18].
- First 3-D structure
- Bacillus subtilis levansucrase .
- KOSHLAND DE Jr and STEIN SS. (1954) Correlation of bond breaking with enzyme specificity; cleavage point of invertase. J Biol Chem. 208, 139-48.
- Chambert R, Treboul G, and Dedonder R. (1974) Kinetic studies of levansucrase of Bacillus subtilis. Eur J Biochem. 41, 285-300. DOI:10.1111/j.1432-1033.1974.tb03269.x |
- 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. 309 ( Pt 1), 113-8. DOI:10.1042/bj3090113 |
- Song DD and Jacques NA. (1999) Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975. Biochem J. 341 ( Pt 2), 285-91.
- Meng G and Fütterer K. (2003) Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Biol. 10, 935-41. DOI:10.1038/nsb974 |
- 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. 132, 565-72. DOI:10.1093/oxfordjournals.jbchem.a003258 |
- Batista FR, Hernández L, Fernández JR, Arrieta J, Menéndez C, Gómez R, Támbara Y, and Pons T. (1999) Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity. Biochem J. 337 ( Pt 3), 503-6.
- Isono N, Tochihara T, Kusnadi Y, Win TT, Watanabe K, Obae K, Ito H, and Matsui H. (2004) Cloning and heterologous expression of a beta-fructofuranosidase gene from Arthrobacter globiformis IFO 3062, and site-directed mutagenesis of the essential aspartic acid and glutamic acid of the active site. J Biosci Bioeng. 97, 244-9. DOI:10.1016/S1389-1723(04)70199-1 |
- Ozimek LK, van Hijum SA, van Koningsveld GA, van Der Maarel MJ, van Geel-Schutten GH, and Dijkhuizen L. (2004) Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121. FEBS Lett. 560, 131-3. DOI:10.1016/S0014-5793(04)00085-7 |
- 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. 390, 19-27. DOI:10.1042/BJ20050324 |
- Strube CP, Homann A, Gamer M, Jahn D, Seibel J, and Heinz DW. (2011) Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs. J Biol Chem. 286, 17593-600. DOI:10.1074/jbc.M110.203166 |
- Pijning T, Anwar MA, Böger M, Dobruchowska JM, Leemhuis H, Kralj S, Dijkhuizen L, and Dijkstra BW. (2011) Crystal structure of inulosucrase from Lactobacillus: insights into the substrate specificity and product specificity of GH68 fructansucrases. J Mol Biol. 412, 80-93. DOI:10.1016/j.jmb.2011.07.031 |
- Tonozuka T, Tamaki A, Yokoi G, Miyazaki T, Ichikawa M, Nishikawa A, Ohta Y, Hidaka Y, Katayama K, Hatada Y, Ito T, and Fujita K. (2012) Crystal structure of a lactosucrose-producing enzyme, Arthrobacter sp. K-1 β-fructofuranosidase. Enzyme Microb Technol. 51, 359-65. DOI:10.1016/j.enzmictec.2012.08.004 |
- Wuerges J, Caputi L, Cianci M, Boivin S, Meijers R, and Benini S. (2015) The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site. J Struct Biol. 191, 290-8. DOI:10.1016/j.jsb.2015.07.010 |
- Naumoff DG (2001) beta-fructosidase superfamily: homology with some alpha-L-arabinases and beta-D-xylosidases. Proteins. 42, 66-76. DOI:10.1002/1097-0134(20010101)42:1<66::aid-prot70>3.0.co;2-4 |
- Pons T, Olmea O, Chinea G, Beldarraín A, Márquez G, Acosta N, Rodríguez L, and Valencia A. (1998) Structural model for family 32 of glycosyl-hydrolase enzymes. Proteins. 33, 383-95. DOI:10.1002/(sici)1097-0134(19981115)33:3<383::aid-prot7>3.0.co;2-r |
- Pons T, Hernández L, Batista FR, and Chinea G. (2000) Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity. Protein Sci. 9, 2285-91. DOI:10.1110/ps.9.11.2285 |
- Chambert R and Gonzy-Tréboul G. (1976) Levansucrase of Bacillus subtilis: kinetic and thermodynamic aspects of transfructosylation processes. Eur J Biochem. 62, 55-64. DOI:10.1111/j.1432-1033.1976.tb10097.x |