Glycoside Hydrolase Family 32

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• Authors: Mirjam Czjzek and Wim Van den Ende
• Responsible Curator: Mirjam Czjzek

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Glycoside Hydrolase Family GH32
Clan	GH-J
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/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 activities 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 GH32 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 transglycosylase 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

The two residues, responsible for the catalytic reaction in family GH32 enzymes, have first been identified in yeast invertase as an aspartate located close to the N-terminus acting as the catalytic nucleophile [3] and a glutamate acting as the general acid/base [4]. An interesting feature concerns some members of GH32, such as the endo-inulinase from Aspergillus ficuum, that use a glutamate replacing the aspartate as catalytic nucleophile [5].

Three-dimensional structures

Several three dimensional structures of family GH32 enzymes have been solved so far. The first crystal structure was reported for the bacterial β-fructosidase from Thermotoga maritima [6]. Further crystal structures of enzymes and their substrate-complexes have been solved for two plant enzymes (cell wall invertase [7] and fructan 1-exohydrolase [8] ), as well as one fungal exo-inulinase [9]. The core of the structure consists of a five-bladed β-propeller appended to a β-sandwich, consisting of two sheets of six β-strands. Although sequence similarity is low within the sandwich modules, all family GH32 members contain such a module. A structural relationship of the catalytic core module exists to family GH68 (also member of Clan GH-J) and family GH43, as predicted by detailed sequence analysis [10]. All three enzyme families display a five bladed β-propeller fold.

Family Firsts

First sterochemistry determination

Saccharomyces cerevisiae invertase [2].

First catalytic nucleophile identification

Saccharomyces cerevisiae invertase [3]

First general acid/base residue identification

Saccharomyces cerevisiae invertase [4]

First 3-D structure

Bacterial β-fructosidase from Thermotoga maritima by X-ray crystallography (PDB ID 1uyp) [6]

References

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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
10. Naumoff DG (2001). beta-fructosidase superfamily: homology with some alpha-L-arabinases and beta-D-xylosidases. Proteins. 2001;42(1):66-76. DOI:10.1002/1097-0134(20010101)42:1<66::aid-prot70>3.0.co;2-4 |