Crystallization and preliminary X-ray diffraction study of fructan 1-exohydrolase IIa from<i>Cichorium intybus</i>

Verhaest, Maureen; Ende, Wim Van den; Yoshida, Midori; Roy, Katrien Le; Peeraer, Yves; Sansen, Stefaan; Ranter, C. J. De; Laere, André Van; Rabijns, A.

doi:10.1107/s0907444903029317

Cited by 11 publications

(10 citation statements)

References 15 publications

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“…Heterologous expression in P. pastoris proved a valuable system to judge the main functionality of fructosyltransferases (Kawakami & Yoshida, 2002) and FEHs (Van den Ende et al ., 2003b; Verhaest et al ., 2004), although sometimes an additional side activity was found compared with the native enzyme (Hochstrasser et al ., 1998). Table 1 shows the extreme substrate selectivity towards 6‐kestose for the heterologously expressed 6‐KEH w1 and w2 enzymes.…”

Section: Resultsmentioning

confidence: 99%

Cloning, characterization and functional analysis of novel 6‐kestose exohydrolases (6‐KEHs) from wheat (Triticum aestivum)

et al. 2005

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Summary• Cereals accumulate graminan-type fructans which are subject to stress-related degradation by fructan 1-exohydrolases (1-FEHs) and fructan 6-exohydrolases (6-FEHs). To find new FEH genes related to freezing tolerance, a cold-hardened wheat crown cDNA library was screened.• Here we report the cloning, purification and characterization of two novel 6-kestosidase (6-KEH) isoenzymes from wheat crowns ( Triticum aestivum ). Functional characterization in Pichia pastoris confirmed the extreme substrate selectivity for the fructan trisaccharide 6-kestose.• Northern blotting showed that 6-KEH transcripts were constantly detected at the same level from autumn to winter in crown but not in leaf tissues. Apoplastic fluid isolations and activity measurements strongly suggest that 6-KEH is localized in the apoplast.• It is proposed that 6-KEHs, together with other FEHs, might be involved in the breakdown of apoplastic fructans which may fulfil a role as membrane protectors under stress. Alternatively, a role in signalling processes, or in the degradation of exogenous 6-kestose from bacterial origin, cannot be excluded.

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Section: Resultsmentioning

confidence: 99%

Cloning, characterization and functional analysis of novel 6‐kestose exohydrolases (6‐KEHs) from wheat (Triticum aestivum)

et al. 2005

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“…As the mutant differed by only one amino acid from the active protein, it could be expected that this mutant would crystallize in a similar condition as the native 1‐FEH IIa (Verhaest et al ., 2004). To test this, a screening was set up, changing only the salt concentration of the native 1‐FEH IIa crystallization condition.…”

Section: Methodsmentioning

confidence: 99%

“…The wild-type and mutant enzymes were purified as described previously (Verhaest et al ., 2004). The protein solution was concentrated to 10 mg ml − 1 by ultrafiltration using a Microcon concentrator (Amicon, Beverly, MA, USA) with a 3 kDa cutoff.…”

Section: Mutagenesis Purification Crystallization and Data Collectionmentioning

confidence: 99%

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

et al. 2007

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Summary Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. Here we report on complexes between chicory (Cichorium intybus) 1‐FEH IIa with the substrate 1‐kestose and the inhibitors sucrose, fructose and 2,5 dideoxy‐2,5‐imino‐D‐mannitol. Comparisons with other family GH32 and 68 enzyme‐substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1‐FEH IIa). Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H‐linkage with the acid‐base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1‐kestose forms an intramolecular H‐linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose‐inhibited FEHs show a Ki (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.

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“…Cichorium intybus L. 1‐FEH IIa (Ci1‐FEHIIa) and B. vulgaris 6‐FEH (Bv6‐FEH) were cloned into the pPICZαA vector as described by Verhaest et al. (2004); Van den Ende et al.…”

Section: Methodsmentioning

confidence: 99%

Influencing the binding configuration of sucrose in the active sites of chicory fructan 1‐exohydrolase and sugar beet fructan 6‐exohydrolase

et al. 2008

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Summary• The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis.• It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration.• In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a β-fructofuranosidase type of enzyme with dual activity against sucrose and levan.• As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.

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Crystallization and preliminary X-ray diffraction study of fructan 1-exohydrolase IIa fromCichorium intybus

Cited by 11 publications

References 15 publications

Cloning, characterization and functional analysis of novel 6‐kestose exohydrolases (6‐KEHs) from wheat (Triticum aestivum)

Cloning, characterization and functional analysis of novel 6‐kestose exohydrolases (6‐KEHs) from wheat (Triticum aestivum)

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

Influencing the binding configuration of sucrose in the active sites of chicory fructan 1‐exohydrolase and sugar beet fructan 6‐exohydrolase

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