Insights into the fine architecture of the active site of chicory fructan 1‐exohydrolase: 1‐kestose as substrate vs sucrose as inhibitor

Verhaest, Maureen; Lammens, Willem; Roy, Katrien Le; Ranter, C. J. De; Laere, André Van; Rabijns, A.; Ende, Wim Van den

doi:10.1111/j.1469-8137.2007.01988.x

Cited by 68 publications

(85 citation statements)

References 46 publications

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“…In contrast, the substrate is loosely bound at subsite ϩ2 or dangling into the solvent. In agreement with this observation, short substrates such as 1-kestose or raffinose are accommodated in essentially the same conformation in all the formerly described complexes (13,16,18,19). In contrast, XdINV presents a deep cavity showing multiple binding sites, a feature that has been previously described for two other fungal enzymes, the AjFT and the SoFfase.…”

Section: Discussionsupporting

confidence: 65%

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity

Ramirez-Escudero

Gimeno-Pérez

González

et al. 2016

Journal of Biological Chemistry

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Xanthophyllomyces dendrorhous ␤-fructofuranosidase (XdINV) is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with an N-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite ؉2 depending on the substrate, and thus, a flexible loop (Glu-334 -His-343) is essential in binding sucrose and ␤(2-1)-linked oligosaccharides. Conversely, ␤(2-6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on ␣-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates.

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Section: Discussionsupporting

confidence: 65%

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity

Ramirez-Escudero

Gimeno-Pérez

González

et al. 2016

Journal of Biological Chemistry

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“…Although the exact position of Suc in the active site of AtcwINV1 is unknown and the structure of the AtcwINV1-Suc complex remains to be determined, the orientation of Suc in AtcwINV1 can be predicted (Fig. 6C) based on the position of Suc in the active site of B. subtilis levansucrase because the position of the terminal fructosyl at the 21 subsite is equal for all GH32 and GH68 enzymes (Verhaest et al, 2007). An Asp-239/Glu-340 functional homolog can also be found in G. diazotrophicus levansucrase (Gln-399) and T. maritima invertase (Glu-188).…”

Section: Resultsmentioning

confidence: 99%

Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase

et al. 2007

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Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.

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“…Therefore, it seems reasonable to assume that during evolution, defective cwINVs were mainly generated by mutating the Asp-239 homolog (as is the case in all FEHs; Van den Ende et al, 2009), allowing the binding of Suc as an inhibitor but not as a substrate, without interfering with fructan degradation (e.g. Suc-inhibited FEHs; Verhaest et al, 2007). Further mutations in the surrounding Trp residues probably led to gradually more inactivation or, as is the case for Nin88, a completely inactivated form.…”

Section: Discussionmentioning

confidence: 99%

Understanding the Role of Defective Invertases in Plants: Tobacco Nin88 Fails to Degrade Sucrose

et al. 2013

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Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs.

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Insights into the fine architecture of the active site of chicory fructan 1‐exohydrolase: 1‐kestose as substrate vs sucrose as inhibitor

Cited by 68 publications

References 46 publications

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity

Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase

Understanding the Role of Defective Invertases in Plants: Tobacco Nin88 Fails to Degrade Sucrose

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