Glycoside Hydrolase Family 32

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• Author: Mirjam Czjzek
• Responsible Curator: Mirjam Czjzek

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Glycoside Hydrolase Family GH32
Clan	GH-J
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH32.html

Substrate specificities

Glycoside hydrolase family GH32 contains one of the earliest described enzyme activities, namely that of 'inverting' sucrose, from which is derived the name of 'invertase' (EC 3.2.1.26), discovered in the second half of the 19th century [1]. Besides the 'historical' invertases, this family also contains enzymes that hydrolyze fructose containing polysaccharides such as inulinases (EC 3.2.1.7) and exo-inulinases (EC 3.2.1.80), levanases (EC 3.2.1.65) and β-2,6-fructan 6-levanbiohydrolases(EC 3.2.1.64), fructan β-(2,1)-fructosidase/1-exohydrolase (EC 3.2.1.153) or fructan β-(2,6)-fructosidase/6-exohydrolases (EC 3.2.1.154), as well as enzymes displaying transglycosylating activites such as sucrose:sucrose 1-fructosyltransferases (EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (EC 2.4.1.100), sucrose:fructan 6-fructosyltransferase (EC 2.4.1.10), fructan:fructan 6G-fructosyltransferase (EC 2.4.1.243) and levan fructosyltransferases (EC 2.4.1.-).

Kinetics and Mechanism

Family 32 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 [2]. The transfructosylation activity (a type of transglycosylation activity) observed for invertase in this reaction indicated that the enzyme operates with a molecular mechanism leading to overall retention of the anomeric configuration [2].

Catalytic Residues

    Normal   0   21                             MicrosoftInternetExplorer4     The two invariant residues, responsible for the catalytic reaction in family 32 enzymes, have first been identified experimentally in yeast invertase as an aspartate located close to the N-terminus acting as the nucleophile [3] and a glutamate acting as the general acid/base [4].

Three-dimensional structures

Several three dimensional structures of family GH32 enzymes have been solved. The first crystal structure was reported for the bacterial β-fructosidase from Thermotoga maritima [5]. Further crystal structures of enzymes and their substrate complexes have been solved for two plant enzymes (cell wall invertase [6] and fructan-1-exohydrolase [7] ), as well as one fungal exo-inulinase [7]. The core of the structure consists of a five-bladed β-propeller appended to a β-sandwich consisting of two sheets of six strands. Although sequence similarity is low within this β-sandwich domain, most invertase are appended to such a sandwich domain. Structural similarity to family GH68 (also belonging to clan GH-J) and

Family Firsts

First sterochemistry determination

Cite some reference here, with a short explanation [1].

First catalytic nucleophile identification

First general acid/base residue identification

First 3-D structure

References

1. O'Sullivan, C., and Tompson, F. W. (1890) J. Chem. Soc. 57, 854-870

2. 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
3. Reddy VA and Maley F. (1990). Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis. J Biol Chem. 1990;265(19):10817-20. | Google Books | Open Library
4. Reddy A and Maley F. (1996). Studies on identifying the catalytic role of Glu-204 in the active site of yeast invertase. J Biol Chem. 1996;271(24):13953-7. DOI:10.1074/jbc.271.24.13953 |
5. Alberto F, Bignon C, Sulzenbacher G, Henrissat B, and Czjzek M. (2004). The three-dimensional structure of invertase (beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases. J Biol Chem. 2004;279(18):18903-10. DOI:10.1074/jbc.M313911200 |
6. Alberto F, Bignon C, Sulzenbacher G, Henrissat B, and Czjzek M. (2004). The three-dimensional structure of invertase (beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases. J Biol Chem. 2004;279(18):18903-10. DOI:10.1074/jbc.M313911200 |
7. Alberto F, Bignon C, Sulzenbacher G, Henrissat B, and Czjzek M. (2004). The three-dimensional structure of invertase (beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases. J Biol Chem. 2004;279(18):18903-10. DOI:10.1074/jbc.M313911200 |