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Title: Structure-function analysis of Glycoside Hydrolase Family 32 (GH32) plant invertases and fructan exohydrolases
Other Titles: Structuur-functie analyse van Glycoside Hydrolase familie 32 (GH32) plant invertasen en fructan exohydrolasen.
Authors: Le Roy, Katrien; M9712659
Issue Date: 29-Oct-2007
Abstract: Structure-function analysis of Glycoside Hydrolase Family 32 (GH32) plant invertases and fructan exohydrolases Fructans are sucrose based polymers of fructose occuring in about 15% of the flowering plants. The degradation of fructans is catalyzed by fructan exohydrolases (FEHs), releasing one terminal fructosyl at a time using water as an acceptor. All FEHs purified so far were found to be unifunctional enzymes being very specific for fructans (Fru-Fru linkage) as donor substrate and unable to degrade sucrose (Glc-Fru linkage). Sucrose is hydrolyzed by invertases. Plant FEHs are closely related to cell wallinvertases at the molecular and structural level and are grouped in theGlycoside Hydrolase Family 32 (GH32). Both FEHs and invertases can in fact be considered as fructosyltransferases only differing in donor substrate specificity. The high levels of sequence similarity between these enzymes reveal an important evolutionary relationship. In this doctoral study, we focussed on three main subjects dealing withthe differences in substrate specificity between the different types ofGH32 family hydrolases. In a first part, the binding and hydrolysis of sucrose in invertases was investigated. Until now, it remained unclear which amino acid residues determined whether sucrose or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. The three dimensional structure determination of Cichorium intybus 1-FEH IIa and Arabidopsis thaliana cell wall invertase 1 (AtcwINV1) was important to gain further insights into the specific differences betweenthese two types of closely related enzymes. Site-directed mutagenesis studies on AtcwINV1 and Beta vulgaris 6-FEH demonstrated an important role for Asp239 and Trp47 in sucrose binding and hydrolysis. Moreover, an AtcwINV1 D239A mutant acted as a FEH preferentially degrading 1-kestose, strongly supporting the general idea that FEHs could have evolved from a cell wall invertase ancestor by few mutational changes. In general, GH32 family enzymes containing an Asp239 functional homologue have sucrose as preferential donor, while enzymes lacking this homologue use fructans as their donor substrate. Asp239 and Trp47 are proposed as reliable determinants for the identification of non characterized members within the group of cell wall invertases/ FEHs. This predictive tool is a rapidway to confirm the invertase functionality of enzymes catalogued as such and can be considered as an important alternative for functional characterization. Secondly, the inhibitory effect of sucrose on FEHs was examined. Sucrose was found to be a strong inhibitor for many, but not all, FEHs including chicory 1-FEH IIa. The three dimensional structure determination of chicory 1-FEH IIa in complex with its preferential substrate 1-kestose and its inhibitor sucrose, provides further insights why sucrose acts as an inhibitor in 1-FEH IIa and not as a substrate. Comparison of several GH32 and GH68 protein structures in complex with their substrates, reveals that the terminal fructosyl unit is always bound in a very similar wayat the -1 subsite. Apparently, the presence of a highly conserved Trp82homologue (WSGSAT region) is crucial for the stabilization of the substrate in the active site, as confirmed by site-directed mutagenesis studies. Interestingly, in chicory 1-FEH IIa, this Trp residue has a different orientation, most probably allowing sucrose to bind in an alternative inhibitor configuration. This different orientation is determined by theabsence of a stacking hydrophobic residue, as observed for all sucrose inhibited FEHs and confirmed by site-directed mutagenesis studies. A final part deals with the binding of inulin and levan in FEHs. Unfortunately, no three dimensional structures of chicory 1-FEH IIa in complexwith higher DP inulin could be obtained. However, site-directed mutagenesis studies on chicory 1-FEH IIa strongly support the presence of an inulin binding cleft between the two structural domains. Introduction of an N-glycosylation site near this cleft most probably prevents inulin binding and degradation. Since the presence of such a glycosyl chain blocking this cleft is highly conserved among 6-FEHs, the binding of levan occurs most probably in a different way and at a different location. Although any structural data on 6-FEHs are lacking so far, modeling studies and some site-directed mutagenesis experiments are indicative for the presence of an alternative levan and 6-kestose binding site. Moreover, the difference in substrate specificity between β, 2-1 and β, 2-6 hydrolyzing FEHs seems to be (partly) determined by the Trp82 residue. In conclusion, this doctoral study provides the first insights into thefundamental differences in substrate specificity between the different types of GH32 hydrolases. Some critical amino acids with respect to sucrose binding, in both substrate and inhibitor configuration, and hydrolysis could be identified. Also some first insights on the difference in substrate specificity between β, 2-1 and β, 2-6 hydrolyzing FEHswere obtained.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Physiology of Plants and Micro-organisms Section - miscellaneous

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