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Glycoside Hydrolase Family 68
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|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 220.127.116.11), β-fructofuranosidase (EC 18.104.22.168), and inulosucrase (EC 22.214.171.124). 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, only five 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 four is an inulosucrase (InuJ) from Lactobacillus johnsonii NCC533 , and the last one corresponds to beta-fructofuranosidase (ArFFase) from Arthrobacter sp. K-1 . 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 [16, 17].
- First 3-D structure
- Bacillus subtilis levansucrase .
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- Chambert R, Treboul G, and Dedonder R. Kinetic studies of levansucrase of Bacillus subtilis. Eur J Biochem. 1974 Jan 16;41(2):285-300.
- Hernandez L, Arrieta J, Menendez C, Vazquez R, Coego A, Suarez V, Selman G, Petit-Glatron MF, and Chambert R. Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochem J. 1995 Jul 1;309 ( Pt 1):113-8.
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- Yanase H, Maeda M, Hagiwara E, Yagi H, Taniguchi K, and Okamoto K. Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase. J Biochem. 2002 Oct;132(4):565-72.
- Batista FR, Hernández L, Fernández JR, Arrieta J, Menéndez C, Gómez R, Támbara Y, and Pons T. 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. 1999 Feb 1;337 ( Pt 3):503-6.
- Isono N, Tochihara T, Kusnadi Y, Win TT, Watanabe K, Obae K, Ito H, and Matsui H. 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. 2004;97(4):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. Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121. FEBS Lett. 2004 Feb 27;560(1-3):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. Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus. Biochem J. 2005 Aug 15;390(Pt 1):19-27. DOI:10.1042/BJ20050324 |
- Strube CP, Homann A, Gamer M, Jahn D, Seibel J, and Heinz DW. Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs. J Biol Chem. 2011 May 20;286(20):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. Crystal structure of inulosucrase from Lactobacillus: insights into the substrate specificity and product specificity of GH68 fructansucrases. J Mol Biol. 2011 Sep 9;412(1):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. Crystal structure of a lactosucrose-producing enzyme, Arthrobacter sp. K-1 β-fructofuranosidase. Enzyme Microb Technol. 2012 Dec 10;51(6-7):359-65. DOI:10.1016/j.enzmictec.2012.08.004 |
- Naumoff DG. beta-fructosidase superfamily: homology with some alpha-L-arabinases and beta-D-xylosidases. Proteins. 2001 Jan 1;42(1):66-76.
- Pons T, Olmea O, Chinea G, Beldarraín A, Márquez G, Acosta N, Rodríguez L, and Valencia A. Structural model for family 32 of glycosyl-hydrolase enzymes. Proteins. 1998 Nov 15;33(3):383-95.
- Pons T, Hernández L, Batista FR, and Chinea G. Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity. Protein Sci. 2000 Nov;9(11):2285-91. DOI:10.1110/ps.9.11.2285 |
- Chambert R and Gonzy-Tréboul G. Levansucrase of Bacillus subtilis: kinetic and thermodynamic aspects of transfructosylation processes. Eur J Biochem. 1976 Feb 2;62(1):55-64.