Gene Cloning and Functional Characterization by Heterologous Expression of the Fructosyltransferase of
            <i>Aspergillus sydowi</i>
            IAM 2544

Heyer, Arnd G.; Wendenburg, Regina

doi:10.1128/aem.67.1.363-370.2001

Cited by 43 publications

(32 citation statements)

References 48 publications

(45 reference statements)

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“…SucA, which is identical to Suc1, is therefore likely to encode a fructofuranosidase lacking any detectable fructosyltransferase activity under the given assay conditions. A second fungal fructosyltransferase-encoding gene was identified in Aspergillus sydowi (sftA) (Heyer & Wendenburg, 2001), which clusters with FopA and SucA (Fig. 1).…”

Section: Discussionmentioning

confidence: 99%

“…Apart from displaying substrate hydrolysis, some of these enzymes can also perform transfructosylation reactions, producing the trisaccharide 1-kestose from sucrose (Rehm et al, 1998;Sangeetha et al, 2004;Yanai et al, 2001) and even longer fructo-oligosaccharides (Heyer & Wendenburg, 2001). Currently, all known fungal inulin-modifying enzymes are grouped together in family 32 of glycoside hydrolases (GH32) (http://afmb.cnrs-mrs.fr/CAZY/index.…”

Section: Introductionmentioning

confidence: 99%

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Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

et al. 2006

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As a soil fungus, Aspergillus niger can metabolize a wide variety of carbon sources, employing sets of enzymes able to degrade plant-derived polysaccharides. In this study the genome sequence of A. niger strain CBS 513.88 was surveyed, to analyse the gene/enzyme network involved in utilization of the plant storage polymer inulin, and of sucrose, the substrate for inulin synthesis in plants. In addition to three known activities, encoded by the genes suc1 (invertase activity; designated sucA), inuE (exo-inulinase activity) and inuA/inuB (endo-inulinase activity), two new putative invertase-like proteins were identified. These two putative proteins lack N-terminal signal sequences and therefore are expected to be intracellular enzymes. One of these two genes, designated sucB, is expressed at a low level, and its expression is up-regulated when A. niger is grown on sucrose-or inulin-containing media. Transcriptional analysis of the genes encoding the sucrose-(sucA) and inulin-hydrolysing enzymes (inuA and inuE) indicated that they are similarly regulated and all strongly induced on sucrose and inulin. Analysis of a DcreA mutant strain of A. niger revealed that expression of the extracellular inulinolytic enzymes is under control of the catabolite repressor CreA. Expression of the inulinolytic enzymes was not induced by fructose, not even in the DcreA background, indicating that fructose did not act as an inducer. Evidence is provided that sucrose, or a sucrose-derived intermediate, but not fructose, acts as an inducer for the expression of inulinolytic genes in A. niger.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

et al. 2006

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“…The FT from Aspergillus possess both hydrolytic and transfructosylating activities. For sucrose at concentration of Ͼ100 mM, FT exhibit almost an exclusive transfructosylation activity (12)(13)(14).…”

mentioning

confidence: 97%

“…Fructosyltransferases (FTs, EC 2.4.1.9) expressed in species of Aspergillus, Penicillium, Aureobasidium, and Kluyveromyces are the most studied fungal FT enzymes (5,(12)(13)(14)(15). Fungal FT act on sucrose by cleaving the ␤-(231) linkage, releasing glucose, and then transferring the fructosyl group to an acceptor molecule.…”

mentioning

confidence: 99%

Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

Chuankhayan

Hsieh

Huang

et al. 2010

Journal of Biological Chemistry

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Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade ␤-propeller fold linked to a C-terminal ␤-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (؊1 to ؉3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the ؊1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the ؉1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the ؉2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the ؉2 and ؉3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.

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“…Fungal invertases are well studied at the biochemical level, especially the enzymes from Saccharomyces cerevisiae and several Aspergillus spp., the latter mainly because of the capability of some invertases to produce fructooligosaccharides at a technical scale (Boddy et al 1993;Heyer and Wendenburg 2001;Neumann and Lampen 1967;Yanai et al 2001). Although the biochemical characteristics are not fundamentally different from the plant enzymes, the DNA sequences indicate a separate evolution of fungal invertases.…”

mentioning

confidence: 99%

Cloning and Characterization of a Novel Invertase from the Obligate Biotroph Uromyces fabae and Analysis of Expression Patterns of Host and Pathogen Invertases in the Course of Infection

Voegele

Wirsel²,

Möll³

et al. 2006

MPMI

100

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Invertases are key enzymes in carbon partitioning in higher plants. They gain additional importance in the distribution of carbohydrates in the event of wounding or pathogen attack. Although many researchers have found an increase in invertase activity upon infection, only a few studies were able to determine whether the source of this activity was host or parasite. This article analyzes the role of invertases involved in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant, Vicia faba. We have identified a fungal gene, Uf-INV1, with homology to invertases and assessed its contribution to pathogenesis. Expression analysis indicated that transcription began upon penetration of the fungus into the leaf, with high expression levels in haustoria. Heterologous expression of Uf-INV1 in Saccharomyces cerevisiae and Pichia pastoris allowed a biochemical characterization of the enzymatic activity associated with the secreted gene product INV1p. Expression analysis of the known vacuolar and cell-wallbound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cellwall-associated invertase exhibited increased expression. These changes were not confined to the infected tissue, and effects also were observed in remote plant organs, such as roots. These findings hint at systemic effects of pathogen infection. Our results support the hypothesis that pathogen infection establishes new sinks which compete with physiological sink organs.

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Gene Cloning and Functional Characterization by Heterologous Expression of the Fructosyltransferase of Aspergillus sydowi IAM 2544

Cited by 43 publications

References 48 publications

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

Cloning and Characterization of a Novel Invertase from the Obligate Biotroph Uromyces fabae and Analysis of Expression Patterns of Host and Pathogen Invertases in the Course of Infection

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