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Glycoside Hydrolase Family 32

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Glycoside Hydrolase Family GH32
Clan GH-J
Mechanism retaining
Active site residues known
CAZy DB link
http://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

  1. O'Sullivan, C., and Tompson, F. W. (1890) J. Chem. Soc. 57, 854-870
    [1]
  2. KOSHLAND DE Jr and STEIN SS. (1954) Correlation of bond breaking with enzyme specificity; cleavage point of invertase. J Biol Chem. 208, 139-48. PubMed ID:13174523 | HubMed [2]
  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. 265, 10817-20. PubMed ID:2113524 | HubMed [3]
  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. 271, 13953-7. PubMed ID:8662946 | HubMed [4]
  5. Park S, Han Y, Kim H, Song S, Uhm TB, and Chae KS. (2003) Trp17 and Glu20 residues in conserved WMN(D/E)PN motif are essential for Aspergillus ficuum endoinulinase (EC 3.2.1.7) activity. Biochemistry (Mosc). 68, 658-61. PubMed ID:12943511 | HubMed [5]
  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. 279, 18903-10. DOI:10.1074/jbc.M313911200 | PubMed ID:14973124 | HubMed [6]
  7. Verhaest M, Lammens W, Le Roy K, De Coninck B, De Ranter CJ, Van Laere A, Van den Ende W, and Rabijns A. (2006) X-ray diffraction structure of a cell-wall invertase from Arabidopsis thaliana. Acta Crystallogr D Biol Crystallogr. 62, 1555-63. DOI:10.1107/S0907444906044489 | PubMed ID:17139091 | HubMed [7]
  8. Verhaest M, Van den Ende W, Roy KL, De Ranter CJ, Laere AV, and Rabijns A. (2005) X-ray diffraction structure of a plant glycosyl hydrolase family 32 protein: fructan 1-exohydrolase IIa of Cichorium intybus. Plant J. 41, 400-11. DOI:10.1111/j.1365-313X.2004.02304.x | PubMed ID:15659099 | HubMed [8]
  9. Nagem RA, Rojas AL, Golubev AM, Korneeva OS, Eneyskaya EV, Kulminskaya AA, Neustroev KN, and Polikarpov I. (2004) Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition. J Mol Biol. 344, 471-80. DOI:10.1016/j.jmb.2004.09.024 | PubMed ID:15522299 | HubMed [9]
  10. Naumoff DG (2001) beta-fructosidase superfamily: homology with some alpha-L-arabinases and beta-D-xylosidases. Proteins. 42, 66-76. PubMed ID:11093261 | HubMed [10]
All Medline abstracts: PubMed | HubMed