Glycoside Hydrolase Family 68

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

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 enzymes include 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). 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 [1]. 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 [2]. Bacterial levansucrases, whatever their origin, catalyze all these reactions but with different efficiency.

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" stabilizes the transition state [5, 7, 8]. The three equivalent acidic residues have been mutated in a β-fructofuranosidase from Arthrobacter globiformis IFO 3062 [9], and in a levansucrase and a inulosucrase from Lactobacillus reuteri 121 [10].

Three-dimensional structures

Currently, only two 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 [6]. The second one corresponds to levansucrase (LdsA) from Gluconacetobacter diazotrophicus SRT4 [11]. 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[12].

Family Firsts

First stereochemistry determination: Bacillus subtilis levansucrase [2].
First catalytic nucleophile identification: Bacillus subtilis levansucrase [6].
First general acid/base residue identification: Zymomonas mobilis levansucrase [7].
First stabilizing transition-state residue identification: Gluconacetobacter diazotrophicus levansucrase [8].
First prediction of a common beta-propeller catalytic domain in GH68 / clan GH-J: Gluconacetobacter diazotrophicus levansucrase [13, 14].
First 3-D structure: Bacillus subtilis levansucrase [6].

References

All Medline abstracts: PubMed